CA2124928C - Hbv amplifier probes for use in solution phase sandwich hybridization assays - Google Patents
Hbv amplifier probes for use in solution phase sandwich hybridization assays Download PDFInfo
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- CA2124928C CA2124928C CA002124928A CA2124928A CA2124928C CA 2124928 C CA2124928 C CA 2124928C CA 002124928 A CA002124928 A CA 002124928A CA 2124928 A CA2124928 A CA 2124928A CA 2124928 C CA2124928 C CA 2124928C
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- HJHVQCXHVMGZNC-JCJNLNMISA-M sodium;(2z)-2-[(3r,4s,5s,8s,9s,10s,11r,13r,14s,16s)-16-acetyloxy-3,11-dihydroxy-4,8,10,14-tetramethyl-2,3,4,5,6,7,9,11,12,13,15,16-dodecahydro-1h-cyclopenta[a]phenanthren-17-ylidene]-6-methylhept-5-enoate Chemical compound [Na+].O[C@@H]([C@@H]12)C[C@H]3\C(=C(/CCC=C(C)C)C([O-])=O)[C@@H](OC(C)=O)C[C@]3(C)[C@@]2(C)CC[C@@H]2[C@]1(C)CC[C@@H](O)[C@H]2C HJHVQCXHVMGZNC-JCJNLNMISA-M 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
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- 229940031000 streptococcus pneumoniae Drugs 0.000 description 1
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- 230000002194 synthesizing effect Effects 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
- C12Q1/706—Specific hybridization probes for hepatitis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/682—Signal amplification
Abstract
Novel DNA probe sequences for detection of HBV in a sample in a solution phase sandwich hybridization assay are de-scribed. Amplified nucleic acid hybridization assays using the probes are exemplified.
Description
WO 93/13120 PCf/US92/11165 HBV AMPLIFIER PROBES FOR USE IN SOLUTION PHASE
SANDWICH HYBRIDIZATION ASSAYS
TP~hnir_gl Field This invention is in the field of nucleic acid hybridization assays. More specifically, it relates to novel nucleic acid probes for detecting Hepatitis B Virus (HBV).
aa~ka_round Art Viral hepatitis is a systemic disease involving primarily the livez, with HBV being primarily responsible for most cases of serum or long-incubation hepatitis.
Antigenic characterization of HHV derives from the complex protein found on the virus's surface. One antigenic specificity, designated a, is common to all HHV
surface antigen (HHSAg), while two other sets of mutually exclusive determinants result in four principle subtypes of HHSAg: adw, ayw, adr, and ayr.
Pasek et al. (Nature 282:575-579, 1979) disclosed the entire nucleotide sequence of subtype ayw HSV genomic DNA. -Valenzuela et al. (An> > Vines Genetics, Field et al., eds., Academic press, NY, 1981) reported the complete nucleotide sequence of subtype adw2 HHV DNA.
EPA Pub. No. 0068719 disclosed the sequence and expression of HHsAg from the adw serotype.
2_ klijiyama et al. (Nucleic Acid Research 11:4601-4610, 1983) disclosed the entire nucleotide sequence of serotype adr HBV DNA.
British patent application No. 2034323A, ' published 6/4/80, describes the isolation and cloning of the HBV genome and its use to detect HBV in serum.
Herninger et al. (.7. Med. Virol. 9:57-68, 1982) discloses an assay based on nucleic acid hybridization which detects and quantitates HHV in serum, using the complete HBV genome as probe.
U.S. 4,562,159 discloses a method and test kit for the detection of HHV by DNA hybridization using cloned, genomic HBV DNA as a probe.
Commonly owned U.S. 4,868,105 describes a solution phase nucleic acid sandwich hybridization assay in which analyte nucleic acid is first hybridized in solution to a labeling probe set and to a capturing probe set in a first vessel. The probe-analyte complex is then transferred to a second vessel that contains a solid-phase-immobilized probe that is substantially complementary to a segment of the capturing probes. The segments hybridize to the immobilized pXObe, thus removing the complex from solution. Having the analyte in the form of an immobilized complex facilitates subsequent separation steps in the assay. Ultimately, single stranded segments of the labeling probe set are hybridized to labeled probes, thus permitting the analyte-containing complex to be detected via a signal generated directly or indirectly from the label.
Commonly owned European Patent Application (EPA) 883096976 discloses a variation in the assay described in U.S. 4,868,105 in which the signal generated by the labeled probes is amplified. The amplification involves the use of nucleic acid multimere. These multimers are branched polynucleotidea that are - 3 - 2i z~92s constructed to have a segment that hybridizes specifically to the analyte nucleic acid or to a nucleic acid (branched or linear) that is bound to the analyze and iterations of a second segment that hybridize specifically to the labeled probe. In the assay employing the multimer, the initial steps of hybridizing the analyte to label or amplifier probe sets and capturing probe sets is a first vessel and transferring the complex to another vessel containing immobilized nucleic acid that will hybridize to a segment of the capturing probes are followed. The multimer is then hybridized to the im~wbilized complex and the labeled ' probes in turn hybridized to the second segment iterations on the multimer. Since the multimers provide a large number of sites for label probe attachment, the signal is amplified. Amplifier and capture probe sequences are disclosed for Hepatitis H virus, Neisseria aonorrhoeag, penicillin and tetracycline- resistance in 1~
non, sad Chlamvdia a ho ~ in.
Commonly owned copending application W092/02526 describes the preparation of large comb-type branched polynucleotide multimers for use in the above-described solution phase assay. The combs provide greater signal enhancement in the assays than the smaller multimers.
Disclosure of the Invention One aspect of the invention is a synthetic oligonucleotide useful as an amplifier probe in a sandwich hybridization assay for IieV comprising a first segment having a nucleotide sequence substantially complementary to a segment of HHV nucleic acid and a second segment having a nucleotide sequence substantially complementary to an oligonucleotide multimer.
._ 212128 Another aspect of the invention is a synthetic oligonucleotide useful as a capture probe in a sandwich hybridization assay for HHV comprising a first segment having a nucleotide sequence substantially complementary .
to a segment of HHV nucleic acid and a second segment having a nucleotide sequence substantially complementary to an oligonucleotide bound to a solid phase.
Another aspect of the invention is a solution sandwich hybridization assay for detecting the presence of HHV in a sample, comprising (a) contacting the sample under hybridizing conditions with an excess of (i) an amplifier probe oligonucleotide comprising a first segment having a nucleotide sequence substantially complementary to a segment of HHV nucleic acid and a second segment having a nucleotide sequence substantially complementary to an oligonucleotide unit of a nucleic acid multimer and (ii) a capture probe oligonucleotide comprising a first segment having a nucleotide sequence that is substantially complementary to a segment of HBV nucleic acid and a second segment that is substantially complementary to an oligonucleotide bound to a solid phase;
(b) contacting the product of step (a) under hybridizing conditions with said oligonucleotide bound to the solid phase;
(c) thereafter separating materials not bound to the solid phase;
(d) contacting the bound product of step (c) under hybridization conditions with the nucleic acid multimer, said multimer comprising at least one oligonucleotide unit that is substantially complementary to the second segment of the amplifier probe polynucleotide and a multiplicity of second ,r WO 93/13120 2 ~. ~ ~ 9 2 g PCT/US92/11165 oligonucleotide units that are substantially complementary to a labeled oligonucleocide;
(e) removing unbound multimez;
(f) contacting under hybridizing conditions the solid phase complex product of step (e) with the labeled oligonucleotide;
(g) removing unbound labeled oligonucleotide;
and (h) detecting the presence of label in the solid phase complex product of step (g).
Another aspect of the invention is a kit for the detection of HHV comprising a kit for the detection of HBV in a sample comprising in combination (i) a net of amplifier probe oligonucleotidea wherein the amplifier probe oligonucleotide comprises a first segment having a nucleotide sequence aubacaatially complementary to a segment of HHV nucleic acid and a second segment having a nucleotide sequence substantially complementary to an oligonucleotide unit of a nucleic acic multimer;
(ii) a set of capture probe oligonucleotides wherein the capture probe oligonucleotide comprises a first segment having a nucleotide sequence that is substantially complementary to a segment of HHV nucleic acid and a second segment that is substantially complementary to an oligonucleotide bound to a solid phase;
(iii) a nucleic acid multimer, said multimer comprising at least one oligonucleotide unit that is substantially complementary to the second segment of the amplifier probe polynucleotide and a multiplicity of second oligonucleotide unite that are aubataatially complementary to a labeled oligonucleotide; and (iv) a labeled oligonucleotide.
- 5a -According to one aspect of the invention there is provided a synthetic oligonucleotide useful as an amplifier probe in a sandwich hybridization assay for Hepatitis B Virus (HBV) comprising a first segment comprising a nucleotide sequence substantially complementary to a segment of HBV
nucleic acid; and a second segment comprising a nucleotide sequence substantially complementary to an oligonucleotide multimer and substantially non-complementary to said segment of HBV nucleic acid;
wherein said nucleotide sequence substantially complementary to a segment of HBV nucleic acid is selected from the group consisting of:
CTGCYCCWT (SEQ ID N0:6), CCTRC1'CGTGTTACAGGCGGGGTZ'lTfCTT (SEQ ID N0:7), TCCATGGCTGCTAGGSTGTRCTGCCAACTG (SEQ ID NO: B), GCYTAYAGACCACCAAATGCCCCTATCYTA (SEQ ID'N0:9), ~TT~C~~~TGCC1TGGGT (SEQ ID N0:10), CATGGAGARCAYMAC11TCAGGATTCCTAGG (SEQ ID N0:11), TCCTGGYTATCGCTGGATGTGTCTGCGGCGT (SEQ ID N0:12), GGCGCTGAATCCYGCGGACGACCCHTCTCG (SEQ ID N0:13), CTTCGCTTCACCTCTGCACGTHGCATGGMG (SHQ ID N0:14), GGTCTSTGCCAAGTGTTTGCTGACGCAACC (SEQ ID N0:15), CCTKCGCGGGACGTCCTTTGTYTACGTCCC (SEQ ID N0:16), MCCTCTGCCfAATCATCTCWTGTWCATGTC (SE~ ID N0:17), CGACCACGGGGCGCACCTCTCTTTACGCGG (SEQ ID NO:lB), TGCCCAAGGTCTTACAYAAGAGGACTCTTG (SEQ ID N0:191, CGTCAATCTYCKCGAGGACTGGGGACCCTG (SEQ ID N0:20), ATGTTGCCCGTTTGTCCfCTAMTTCCAGGA (SEQ ID N0:21), ATCTTC'1'TRTIGG1TCTTCTGGAYTAYCAA (SEQ ID N0:22), ATCATM1'fCCTCT1'CATCCTGCTGCTATGC (SEQ ID N0:23), CAATCACTCACCAACCTCYTGTCCTCCAAY (SEQ ID N0:24), GTGTCYTGGCCAAAATTCGCAGTCCCCAAC (SHQ ID N0:25), CTCG2iGGTGGACITCfC'TCAATTTTCTAGG (SEQ ID N0:26), - 5b - 2124928 GACAAGAATCCTCAC31ATACCRCAGAGTCT (SEQID N0:27), TTTTGGGGTGGAGCCCKCAGGCTCAGGGCR (SEQWD N0:28), CACCATATTCTTGGGAACAAGARCTACAGC (SEQID N0:29), ACACTTCCGGARACTACTGTTGTTAGACGA (SEQID NQ:30), GTVTCTTTYGGAGTG1GGATTCGCACTCCT (SEQID N0:31), TTGGAGCWWC1GTGGAGTTACTCTCRTTIT (SEQID N0:32), ~~CATGGA~TYGAYCCRTATAAAG (SEQID N0:33), AAWGRTCTT1GTAYTAGGAGGCTGTAGGCA (SEQID N0:34), RGACTGGGAGGAGY2GGGGGAGGAGATTAG (SEQID N0:35), CCTTGAGGCMTACTfCAAAGACTGTRTGTT (SEQID N0:36), GTCTGTGCCTTCTCATCTGCCGGWCCG1GT (SEQID N0:37), AGCMGC1TGTT1'TGCTCGCAGSt~GTCIiGG (SEQID N0:38), GGCTCSTCDGCCGATCCATACTGCGGAACT (SEQID N0:39), MTKAACCTTTACCCCG1TGCTCGGCAACGG (SEQID N0:40), GTGGCTCCAGTTCMGGAACAGTAAACCCTG (SEQID N0:41), , RAARCAGGCTTTYACTTTCTCGCCAACTTA (SEQID N0:42), CCTCCRCCTGCCTCYACCAATCGSCAGTCA (SEQID N0:43), ACCAAIITfCI'TYTG'1~TYTGGGTATACAT (SEQID N0:44).
According to another aspect of the invention there is provided a synthetic oligonucleotide useful as a capture probe in a'saadwich hybridization assay for HBv comprising a first segment comprising a nucleotide sequence substantially complementary to a segment of FD3V
nucleic acid; and a second segment eamprising a nucleotide sequence aubetantially complementary to an oligonucleotide bound to a solid phase and substantially non-complementary to said segment of HBV nucleic acid, wherein said nucleotide sequence substantially complementary to a segment of HBV nucleic acid is Selected from the group consisting of:
TATTCCCATCCCATCRTCC1GGGCTTTCGS (SEQ ID N0:45), TATATGGATGATG1GG1'AT1GGGGGCCAAG (SEQ ID N0:46), CGTAGGGCTTTCCCCCACTGTTTGGCTTTC (SEQ ID N0:47), GCTCAGTTTAC~fAGIGCCATTTGTTCAGTG (SEQ ID NOa48), CCTATGGGAGRGGGCCTCAGYCCGTTTCTC (SEQ N0:49), ID
GTCCCCTAGAAGAAGAACTCCCTCGCCTCG (SEQ NO:50), ID
ACGMAGRTCTCMATCGCCGCGTCGCAGAAGA (SEQ
ID
NO:51), CAATCTCGGGAATCTCAATGTTAGTATYCC (SEQ N0:52), ID
GACTCATAAGGTSGGRAACTTTACRGGGCT (SEQ N0:53).
ID
According to a further aspect of the invention there is provided a set of synthetic oligonucleotides useful as amplifier probes in a sandwich hybridization assay for HBV, comprising two oligonucleotides, wherein each member of the set comprises a first segment comprising a nucleotide sequence substantially complementary to a segment of E03V
nucleic acid; and a second segment comprising a nucleotide sequence substantially complementary to an oligonucleotide multimer and substantially non-complementary to said segment of HBV nucleic acid, wherein said nucleotide sequence substantially complementary to a segment of HBV nucleic acid is selected from the group consisting of:
TTGTGGGTCITrfGGGYTTTGCTGCYCCWT (SEQIDN0:6), CCTRCTCGTGTTACAGGCGGGGTiITTCIT (SEQIDN0:7), TCCATGGCTGCTAGGS1GTRC'1'GCCAAC1G (SEQIDN0:8), GCYTAYAGACCACCAAATGCCCC'TATCYTA (SEQIDN0:9), CTGTTCAAGCCTCCAAGCTGTGCCTTGGGT (SEQIDN0:10), CATGGAGARCAYMACATCAGGATTCCTAGG (SEQIDN0:11), TCCTGGYTATCGCTC,GA1'GTGTCTGCGGCGT (SEQ
ID
N0:12), GGCGCTGAATCCYGCGGACGACCCHTCTCG (SEQIDN0:13), CTTCGCTTCACCTCfGCACGTHGCATGGMG (SEQIDN0:14), - Sd - 2124928 GGTCTS1GCCAAG1GTTIGC1GACGCAACC (SEQID NO:15), CCTKCGCGGGACGTCCT1TGTYTACGTCCC (SEQID N0:16), MCCTCTGCCTAATCATCTCWIGTWCATGTC (SEQID N0:17), CGACCACGGGGCGCACCTCTCT1TACGCGG (SEQID NOslH), TGCCCAAGGTCTTACAYAAGAGGACfCTTG (SEQID N0:19), CGTCAATCTSCCRCGAGGACTGGGGACCCTG (SEQID N0:20), ATGTTGCCCGTTTGTCCTCTAM1TCCAGGA (SEQID N0:21), AT~~~T~GTfCTTCfGGAYTAYCAA (SEQID N0:22), ATCATM11'CCTCTTC11TCCTGC1GCTATGC (SHQID N0:23), CAATCACTCACCAACCTCYTGTCCTCCAAY (SEQID N0:24), GTGTCYTGGCCAAAATTCGCAGTCCCCAAC (SEQID N0:25), CTCGTGGTGGAC1TCTCTC31AT1TTCTAGG (SEQID N0:26), GACAAGAATCCTCACAATACCRCAGAGTCT (SEQID N0:27), TTTTGGGGTGGAGCCCRCAGGCT'CAGGGCR (SEQID N0:28), CACCATATTCTTGGGAACAAGARCTACAGC (SEQID N0:29), ACAC2TCCGGARACTACTGTTGTTAGACGA (SEQID N0:30), GTVTC1'fT7CGGAGTGTGGATTCGCACTCCT (SHQID N0:31), TTGGAGCWWCTGTGGAGTTAC1Y.TCRTtTf (SEQID N0:32), TTTGGGGCATGGACATYGAYCCRTATAAAG (SHQID N0:33), AAWGRTCTTTGTAYTAGGAGGCiGTAGGCA (SEQID N0:34), RGACTGGGAGGAGYTGGGGGAGGAGATTAG (SEQID N0:35), CCTTGAGGCMTACTTCAAAGACTGTICTGTT (SEQID N0:36), GTCTGTGCCTTCTCATC1GCCGGWCCG1GT (SEQID N0:37), AGCMGCTTGTTTTGCTCGCAGSMGGTCTGG (SEQID N0:38), GGCTCSTCTGCCGATCCATACTGCGGAACT (SEQID N0:39), htfKAACCTtfACCCCGTTGCTCGGCAACGG (SEQID N0:40), GTGGCTCCAGTTCMGGAACAGTAAACCCTG (SEQID N0:41), RAARCAGGCITf7CACTTTCTCGCCAAC'i'fA (SEQID N0:42) , CCTCCRCC2GCCTCYACCAATCGSCAGTCA (SEQID N0:43), ACCAATTTTCTTYTGTCTYTGGGTATACAT (SHQID N0:44).
According to yet another aspect of the invention there is provided a set of synthetic oligonucleotides useful as capture probes in a sandwich hybridization assay for HBV, comprising two oligonucleotides, wherein each member of the set comprises a first ae9ment comprising a nucleotide sequence substantially complementazy to a segment of HBV
nucleic acid; and 5e a second segment comprising a nucleotide sequence substantially complementary to an oligonucleotide bound to a solid phase and substantially non-complementary to said segment of HBV
nucleic acid, wherein said nucleotide sequence substantially complementary to a segment of HBV nucleic acid is selected from the group consisting of:
TATTCCCATCCCATC&TCCTGGGCTT'i'CGS ( SEQ ID NO ;.4 5 ) , .TATATGGATGA'IGTGGTATt'GGGGGCCAAG (SEQ ID N0:46), CGTAGGGCTTTCCCCCACZGTTT'GGCTTTC (SEQ ID N0:4~), GCTCAGTTTACTAGZGCCATZ'IGTT~UGTG (SEQ ID N0:48) , CCTATC'~GGA~GKG~C~C~Ct~TCAGYCCGTTTGTC (SEQ ID N0:49I , GTCCCCTAGA~1G~LAGAACTCCCI'C't;CCTCG ( S EQ ID NO : 5 0 ) , ACGI~GRTC'rC~ITOGCCGCGTCG~AAGA (SEQ ID NO:51) , CAATGTCGGGAATCTCAATGZ'rAGTATYCC (SEQ ID N0:52), 6ACTCATAAGGTSGGRAAC1'ITAC1CGGGCT (SEQ ID NO: 53 ) .
According to another aspect of the invention there is provided a solution sandwich hybridization assay for detecting the presence of HBV in a sample, comprising (a) contacting the sample under conditions of specific hybridization with an excess of (i) a set of synthetic oligonucleotides useful as amplifier probes in a sandwich hybridization assay for HBV
and (ii) a set of capture probe oligonucleotides wherein the capture probe oligonucleotide comprises a first segment comprising a nucleotide sequence that is substantially complementary to a segment of HBV nucleic acid and a second segment that is substantially complementary to an oligonucleotide bound to a solid phase and substantially non-complementary to said segment of HBV nucleic acid;
(b) contacting the product of step (a) under hybridizing conditions with said oligonucleotide bound to the solid phase;
5f (c) thereafter separating materials not bound to the solid phase;
(d) contacting the bound product of step (c) under hybridization conditions with a nucleic acid multimer, said multimer comprising at least one oligonucleotide unit that is substantially complementary to the second segment of the amplifier probe polynucleotide and a multiplicity of second oligonucleotide units that are substantially complementary to a labeled oligonucleotide;
(e) removing unbound multimer;
(f) contacting under hybridizing conditions the solid phase complex product of step (e) with the labeled oligonucleotide;
(g) removing unbound labeled oligonucleotide; and (h) detecting the presence of label in the solid phase complex product of step (g).
According to another aspect of the invention there is provided a solution sandwich hybridization assay for detecting the presence of HBV in a sample, comprising (a) contacting the sample under conditions of specific hybridization with an excess of (i) a set of amplifier probe oligonucleotides wherein the amplifier probe oligonucleotide comprises a first segment comprising a nucleotide sequence substantially complementary to a segment of HBV nucleic acid and a second segment comprising a nucleotide sequence substantially complementary to an oligonucleotide unit of a nucleic acid multimer and (ii) a set of synthetic oligonucleotides useful as capture probes in a sandwich hybridization assay for HBV;
(b) contacting the product of step (a) under hybridizing conditions with said oligonucleotide bound to the solid phase;
- Sg - 212492a (c) thereafter separating materials not bound to the solid phase;
(d) contacting the bound product of step (c) under hybridization conditions with a nucleic acid auiltimer. said multimer comprising at least one oligonucleotide unit that is substantially complementary to the second segment of the amplifier probe polynucleotide and a multiplicity of second oligonucleotide units that are substantially complementary to a labeled oligonucleotide;
(e) removing unbound multimer;
(f) contacting under hybridizing conditions the solid phase complex product of step (e) with the labeled oligonucleotide;
(g) removing unbound labeled oliqonucleotide;
and (h) detecting Che presence of label in the solid phase complex product of step (g).
According to another aspect of the invention there is provided a kit for the detection of HBV in a sample comprising in combination (i) a set of amplifier probe oligonucleotides wherein the amplifier probe oligonucleotide comprises a first segment comprising a nucleotide sequence substantially complementary to a segment of HHV nucleic acid and a second segment comprising a nucleotide sequence substantially complementary to an oligonucleotide unit of a nucleic acid multimer and substantially non-complementary to said segment of HBV
nucleic acid, (ii) a set of capture probe oligonucleotides wherein the capture probe oligonucleotide comprises a first segment comprising a nucleotide sequence that is substantially complementary to a segment of FD3V DNA sad a second segment that is substantially complementary to an oligonucleotide bound to a solid phase;
- Sh - z~ z49z~
(iii) a nucleic acid multimer, said multimer , comprising at least one oligonucleotide unit that is substantially complemenEary to the second segment of the amplifier probe polynucleotide and a multiplicity of aecoad oligonucleotide units that are subetantialiy complementary to a labeled oligonucleotide; and (iv) a labeled oligoaucleotide.
z~ z~9za These and other embodiments will readily occur to those of ordinary skill is view of the disclosure herein.
' , Modes for Carzvina out the Invention Definitions In defining the present invention, the following terms will be employed, and are intended to be defined as indicated below.
~SOiutioa phase nucleic acid hybridization assay" intends the assay techniques described and claimed in commonly owned U.S. Patent No: 4,868,105 sad EPA
883096976.
A "modified nucleotide' intends a nucleotide monomer that may be atably incorporated into a polyaucleotide and which has an additional functional group. Preferably, the modified nucleotide is a 5'-cytidine is which the N4-position is modified to provide a functional hydroxy group.
An 'amplifier multimer~ intends a branched polynucleotide that is capable of hybridizing simultaneously directly or indirectly to analyte nucleic acid and to a multiplicity of polyhucleotide iterations (i.e., either iterations of another multimer or iterations of a labeled probe). The branching in the multimers is effected through covalent bonds and the multimers are'composed of two types of oligonucleotide unite that are capable of hybridizing, respectively, to analyte nucleic acid or nucleic acid hybridized to analyte nucleic acid and to a multiplicity of labeled probes. The composition and preparation of such multimers are described in EPA 883096976 and W092/02526.
The term "amplifier probe" is intended as a branched or linear polynucleotide that is constructed to 7_ hav= a segment that hybridizes specifically to the ana~~ nucleic acid and iterations of a second segment that h_bridize specifically to an amplifier multimer.
The term ~captuze probe~ is intended as an oligonucleotide having a segment substantially complementary to a nucleotide sequence of the target DNA
and a segment that is substantially complementary to a nucleotide sequence of a solid-phase-immobilized probe.
~Large' as used herein to describe the comb-type branched polynucleotides of the invention intends a molecule having at least about 15 branch sites and at least about 20 iterations of the labeled probe binding sequence.
'Comb-type' as used herein to describe the structure of the branched polynucleotldea of the invention intends a polynucleotide having a linear backbone with a multiplicity of sidechaina extending from the backbone.
A "cleavable linker molecule" intends a molecule that may be atably incorporated into a polynucleotide chain and w_:ich includes a covalent bond that may be broken or cleaved by chemical treatment or physical treatment such as by irradiation.
All nucleic acid sequences disclosed herein are written in a 5' to 3' direction unless otherwise indicated. Nucleotides are designated according to the nucleotide symbols recommended by the IUPAC-IUB
Biochemical Nomenclature.
The general protocol for the solution phase sandwich hybridizations is as follows. The analyte nucleic acid is placed in a microtiter well with an excess of two single-stranded nucleic acid probe seta:
(1) a set of capture probes, each having a first binding WO 93/13t20 PCT/US92/11165 sequence substantially complementary to the analyze and a second binding sequence that is substantially complementary to nucleic acid bound to a solid support, for example, the well surface or a bead, and (2) a set of S amplifier probes (branched or linear), each having a first binding sequence that is capable of specific binding to the analyte and a second binding sequence that is capable of specific binding to a segment of the multimer. The resulting product is a three component nucleic acid complex of the two probes hybridized to the analyze by their first binding sequences. The second binding sequences of the probes regain as single-stranded segments as they are not complementary to the analyte.
This complex hybridizes to the immobilized probe on the solid surface via the second binding sequence of the capture probe. The resulting product comprises the complex bound to the solid surface via the duplex formed by the oligonucleotide bound to the solid surface sad the second binding sequence of the capture probe. Unbound materials are then removed from the surface such as by washing.
The amplification multimer is then added to the bound complex under hybridization conditions to permit the multimer to hybridize to the available second binding sequences) of the amplifier probe of the complex. The resulting complex is then separated from any unbound multimer by washing. The labeled oligonucleotide is then added under conditions which permit it to hybridize to the substantially complementary oligonucleotide units of the multimer. The resulting immobilized labeled nucleic acid complex is then washed to remove unbound labeled oligonucleotide, and read.
The analyte nucleic acids may be from a variety of sources, e.g., biological fluids or solids, and may be prepared for the hybridization analysis by a variety of WO 93/13120 ~ 1 ~ 4 9 2 8 P~/US92/11165 9_ means, e.g., proteinase K/SDS, chaotropic salts, etc.
Also, it may be of advantage to decrease the average size of the analyte nucleic acids by enzymatic, physical or chemical means, e.g., restriction enzymes, sonication, chemical degradation (e.g., metal ions), etc. The fragments may be as small as 0.1 kb, usually being at least about 0.5 kb and may be 1 kb or higher. The analyte sequence is provided in single-stranded form for analysis. Where the sequence is naturally present in single-stranded form, denaturation will not be required.
However, where the sequence may be present in double-stranded form, the sequence should be denatured.
Denaturation can be carried out by various techniques, such as alkali, generally from about 0.05 to 0.2 M
hydroxide, formamide, salts, heat, enzymes, or combinations thereof.
The first binding sequences of the capture probe and amplifier probe that are substantially complementary to the analyte sequence will each be of at least 15 nucleotides, usually at least 25 nucleotides, and not more than about 5 kb, usually not more than about 1 kb, preferably not more than about 100 nucleotides.
They will typically be approximately 30 nucleotides.
They will normally be chosen to bind to different sequences of the analyte. The first binding sequences may be selected based on a variety of considerations.
Depending upon the nature of the analyte, one may be interested in a consensus sequence, a aequeace associated with polymorphisma, a particular phenotype or genotype, a particular strain, or the like.
The number of different amplifier and capture probes used influences the sensitivity of the assay, because the more probe sequences used, the greater the signal provided by the assay system. Furthermore, the use of more probe sequences allows the use of more WO 93/13120 1 2 ~ g 2 g PCT/US92/11165 stringent hybridization conditions, thereby reducing the incidence of false positive results. Thus, the number of probes in a set will be at least one capture probe and at least one amplifier probe, more preferably two capture and two amplifier probes, and most preferably 5-100 capture probes and 5-100 amplifier probes.
Probes for HHV were designed as follows.
EPA 88309676 discloses a set of HBV probes designed by comparing the DNA sequences of the nine HHV subtypes reported in GenBank. Subsequent experimental analysis has demonstrated that these probes were complementary to the subgenomic strand (i.e. plus sense) of the incompletely double-stranded region of HHV, and thus different subsets of these probes hybridized to different viruses, since the length of the subgenomic strands varies among strains. Accordingly, the probe set has been redesigned to comprise sequences aubatantially complementary to the genomic-length strand (i.e, minus-sense) of HHV and to contain fewer spacer regions so as to include more oligonucleotides in the probe set, thereby increasing the sensitivity of the assay system.
In general, regions of greatest homology between the HBV isolates were selected as capture probes, while regions of lesser homology were selected as amplifier probes. Thus, as additional strains or isolates of HBV are made available, appropriate probes made be designed by aligning the sequence of the new strain or isolate with the nucleotide sequences used to design the probes of the present invention, and choosing regions of greatest homology for use as capture probes, with regions of lesser homology chosen ae amplifier probes. The set of presently preferred probes and their capture or amplifier overhang regions, i.e., the regions which hybridize to sequences immobilized on solid support or to an amplifier multimer, are listed in the examples.
SANDWICH HYBRIDIZATION ASSAYS
TP~hnir_gl Field This invention is in the field of nucleic acid hybridization assays. More specifically, it relates to novel nucleic acid probes for detecting Hepatitis B Virus (HBV).
aa~ka_round Art Viral hepatitis is a systemic disease involving primarily the livez, with HBV being primarily responsible for most cases of serum or long-incubation hepatitis.
Antigenic characterization of HHV derives from the complex protein found on the virus's surface. One antigenic specificity, designated a, is common to all HHV
surface antigen (HHSAg), while two other sets of mutually exclusive determinants result in four principle subtypes of HHSAg: adw, ayw, adr, and ayr.
Pasek et al. (Nature 282:575-579, 1979) disclosed the entire nucleotide sequence of subtype ayw HSV genomic DNA. -Valenzuela et al. (An> > Vines Genetics, Field et al., eds., Academic press, NY, 1981) reported the complete nucleotide sequence of subtype adw2 HHV DNA.
EPA Pub. No. 0068719 disclosed the sequence and expression of HHsAg from the adw serotype.
2_ klijiyama et al. (Nucleic Acid Research 11:4601-4610, 1983) disclosed the entire nucleotide sequence of serotype adr HBV DNA.
British patent application No. 2034323A, ' published 6/4/80, describes the isolation and cloning of the HBV genome and its use to detect HBV in serum.
Herninger et al. (.7. Med. Virol. 9:57-68, 1982) discloses an assay based on nucleic acid hybridization which detects and quantitates HHV in serum, using the complete HBV genome as probe.
U.S. 4,562,159 discloses a method and test kit for the detection of HHV by DNA hybridization using cloned, genomic HBV DNA as a probe.
Commonly owned U.S. 4,868,105 describes a solution phase nucleic acid sandwich hybridization assay in which analyte nucleic acid is first hybridized in solution to a labeling probe set and to a capturing probe set in a first vessel. The probe-analyte complex is then transferred to a second vessel that contains a solid-phase-immobilized probe that is substantially complementary to a segment of the capturing probes. The segments hybridize to the immobilized pXObe, thus removing the complex from solution. Having the analyte in the form of an immobilized complex facilitates subsequent separation steps in the assay. Ultimately, single stranded segments of the labeling probe set are hybridized to labeled probes, thus permitting the analyte-containing complex to be detected via a signal generated directly or indirectly from the label.
Commonly owned European Patent Application (EPA) 883096976 discloses a variation in the assay described in U.S. 4,868,105 in which the signal generated by the labeled probes is amplified. The amplification involves the use of nucleic acid multimere. These multimers are branched polynucleotidea that are - 3 - 2i z~92s constructed to have a segment that hybridizes specifically to the analyte nucleic acid or to a nucleic acid (branched or linear) that is bound to the analyze and iterations of a second segment that hybridize specifically to the labeled probe. In the assay employing the multimer, the initial steps of hybridizing the analyte to label or amplifier probe sets and capturing probe sets is a first vessel and transferring the complex to another vessel containing immobilized nucleic acid that will hybridize to a segment of the capturing probes are followed. The multimer is then hybridized to the im~wbilized complex and the labeled ' probes in turn hybridized to the second segment iterations on the multimer. Since the multimers provide a large number of sites for label probe attachment, the signal is amplified. Amplifier and capture probe sequences are disclosed for Hepatitis H virus, Neisseria aonorrhoeag, penicillin and tetracycline- resistance in 1~
non, sad Chlamvdia a ho ~ in.
Commonly owned copending application W092/02526 describes the preparation of large comb-type branched polynucleotide multimers for use in the above-described solution phase assay. The combs provide greater signal enhancement in the assays than the smaller multimers.
Disclosure of the Invention One aspect of the invention is a synthetic oligonucleotide useful as an amplifier probe in a sandwich hybridization assay for IieV comprising a first segment having a nucleotide sequence substantially complementary to a segment of HHV nucleic acid and a second segment having a nucleotide sequence substantially complementary to an oligonucleotide multimer.
._ 212128 Another aspect of the invention is a synthetic oligonucleotide useful as a capture probe in a sandwich hybridization assay for HHV comprising a first segment having a nucleotide sequence substantially complementary .
to a segment of HHV nucleic acid and a second segment having a nucleotide sequence substantially complementary to an oligonucleotide bound to a solid phase.
Another aspect of the invention is a solution sandwich hybridization assay for detecting the presence of HHV in a sample, comprising (a) contacting the sample under hybridizing conditions with an excess of (i) an amplifier probe oligonucleotide comprising a first segment having a nucleotide sequence substantially complementary to a segment of HHV nucleic acid and a second segment having a nucleotide sequence substantially complementary to an oligonucleotide unit of a nucleic acid multimer and (ii) a capture probe oligonucleotide comprising a first segment having a nucleotide sequence that is substantially complementary to a segment of HBV nucleic acid and a second segment that is substantially complementary to an oligonucleotide bound to a solid phase;
(b) contacting the product of step (a) under hybridizing conditions with said oligonucleotide bound to the solid phase;
(c) thereafter separating materials not bound to the solid phase;
(d) contacting the bound product of step (c) under hybridization conditions with the nucleic acid multimer, said multimer comprising at least one oligonucleotide unit that is substantially complementary to the second segment of the amplifier probe polynucleotide and a multiplicity of second ,r WO 93/13120 2 ~. ~ ~ 9 2 g PCT/US92/11165 oligonucleotide units that are substantially complementary to a labeled oligonucleocide;
(e) removing unbound multimez;
(f) contacting under hybridizing conditions the solid phase complex product of step (e) with the labeled oligonucleotide;
(g) removing unbound labeled oligonucleotide;
and (h) detecting the presence of label in the solid phase complex product of step (g).
Another aspect of the invention is a kit for the detection of HHV comprising a kit for the detection of HBV in a sample comprising in combination (i) a net of amplifier probe oligonucleotidea wherein the amplifier probe oligonucleotide comprises a first segment having a nucleotide sequence aubacaatially complementary to a segment of HHV nucleic acid and a second segment having a nucleotide sequence substantially complementary to an oligonucleotide unit of a nucleic acic multimer;
(ii) a set of capture probe oligonucleotides wherein the capture probe oligonucleotide comprises a first segment having a nucleotide sequence that is substantially complementary to a segment of HHV nucleic acid and a second segment that is substantially complementary to an oligonucleotide bound to a solid phase;
(iii) a nucleic acid multimer, said multimer comprising at least one oligonucleotide unit that is substantially complementary to the second segment of the amplifier probe polynucleotide and a multiplicity of second oligonucleotide unite that are aubataatially complementary to a labeled oligonucleotide; and (iv) a labeled oligonucleotide.
- 5a -According to one aspect of the invention there is provided a synthetic oligonucleotide useful as an amplifier probe in a sandwich hybridization assay for Hepatitis B Virus (HBV) comprising a first segment comprising a nucleotide sequence substantially complementary to a segment of HBV
nucleic acid; and a second segment comprising a nucleotide sequence substantially complementary to an oligonucleotide multimer and substantially non-complementary to said segment of HBV nucleic acid;
wherein said nucleotide sequence substantially complementary to a segment of HBV nucleic acid is selected from the group consisting of:
CTGCYCCWT (SEQ ID N0:6), CCTRC1'CGTGTTACAGGCGGGGTZ'lTfCTT (SEQ ID N0:7), TCCATGGCTGCTAGGSTGTRCTGCCAACTG (SEQ ID NO: B), GCYTAYAGACCACCAAATGCCCCTATCYTA (SEQ ID'N0:9), ~TT~C~~~TGCC1TGGGT (SEQ ID N0:10), CATGGAGARCAYMAC11TCAGGATTCCTAGG (SEQ ID N0:11), TCCTGGYTATCGCTGGATGTGTCTGCGGCGT (SEQ ID N0:12), GGCGCTGAATCCYGCGGACGACCCHTCTCG (SEQ ID N0:13), CTTCGCTTCACCTCTGCACGTHGCATGGMG (SHQ ID N0:14), GGTCTSTGCCAAGTGTTTGCTGACGCAACC (SEQ ID N0:15), CCTKCGCGGGACGTCCTTTGTYTACGTCCC (SEQ ID N0:16), MCCTCTGCCfAATCATCTCWTGTWCATGTC (SE~ ID N0:17), CGACCACGGGGCGCACCTCTCTTTACGCGG (SEQ ID NO:lB), TGCCCAAGGTCTTACAYAAGAGGACTCTTG (SEQ ID N0:191, CGTCAATCTYCKCGAGGACTGGGGACCCTG (SEQ ID N0:20), ATGTTGCCCGTTTGTCCfCTAMTTCCAGGA (SEQ ID N0:21), ATCTTC'1'TRTIGG1TCTTCTGGAYTAYCAA (SEQ ID N0:22), ATCATM1'fCCTCT1'CATCCTGCTGCTATGC (SEQ ID N0:23), CAATCACTCACCAACCTCYTGTCCTCCAAY (SEQ ID N0:24), GTGTCYTGGCCAAAATTCGCAGTCCCCAAC (SHQ ID N0:25), CTCG2iGGTGGACITCfC'TCAATTTTCTAGG (SEQ ID N0:26), - 5b - 2124928 GACAAGAATCCTCAC31ATACCRCAGAGTCT (SEQID N0:27), TTTTGGGGTGGAGCCCKCAGGCTCAGGGCR (SEQWD N0:28), CACCATATTCTTGGGAACAAGARCTACAGC (SEQID N0:29), ACACTTCCGGARACTACTGTTGTTAGACGA (SEQID NQ:30), GTVTCTTTYGGAGTG1GGATTCGCACTCCT (SEQID N0:31), TTGGAGCWWC1GTGGAGTTACTCTCRTTIT (SEQID N0:32), ~~CATGGA~TYGAYCCRTATAAAG (SEQID N0:33), AAWGRTCTT1GTAYTAGGAGGCTGTAGGCA (SEQID N0:34), RGACTGGGAGGAGY2GGGGGAGGAGATTAG (SEQID N0:35), CCTTGAGGCMTACTfCAAAGACTGTRTGTT (SEQID N0:36), GTCTGTGCCTTCTCATCTGCCGGWCCG1GT (SEQID N0:37), AGCMGC1TGTT1'TGCTCGCAGSt~GTCIiGG (SEQID N0:38), GGCTCSTCDGCCGATCCATACTGCGGAACT (SEQID N0:39), MTKAACCTTTACCCCG1TGCTCGGCAACGG (SEQID N0:40), GTGGCTCCAGTTCMGGAACAGTAAACCCTG (SEQID N0:41), , RAARCAGGCTTTYACTTTCTCGCCAACTTA (SEQID N0:42), CCTCCRCCTGCCTCYACCAATCGSCAGTCA (SEQID N0:43), ACCAAIITfCI'TYTG'1~TYTGGGTATACAT (SEQID N0:44).
According to another aspect of the invention there is provided a synthetic oligonucleotide useful as a capture probe in a'saadwich hybridization assay for HBv comprising a first segment comprising a nucleotide sequence substantially complementary to a segment of FD3V
nucleic acid; and a second segment eamprising a nucleotide sequence aubetantially complementary to an oligonucleotide bound to a solid phase and substantially non-complementary to said segment of HBV nucleic acid, wherein said nucleotide sequence substantially complementary to a segment of HBV nucleic acid is Selected from the group consisting of:
TATTCCCATCCCATCRTCC1GGGCTTTCGS (SEQ ID N0:45), TATATGGATGATG1GG1'AT1GGGGGCCAAG (SEQ ID N0:46), CGTAGGGCTTTCCCCCACTGTTTGGCTTTC (SEQ ID N0:47), GCTCAGTTTAC~fAGIGCCATTTGTTCAGTG (SEQ ID NOa48), CCTATGGGAGRGGGCCTCAGYCCGTTTCTC (SEQ N0:49), ID
GTCCCCTAGAAGAAGAACTCCCTCGCCTCG (SEQ NO:50), ID
ACGMAGRTCTCMATCGCCGCGTCGCAGAAGA (SEQ
ID
NO:51), CAATCTCGGGAATCTCAATGTTAGTATYCC (SEQ N0:52), ID
GACTCATAAGGTSGGRAACTTTACRGGGCT (SEQ N0:53).
ID
According to a further aspect of the invention there is provided a set of synthetic oligonucleotides useful as amplifier probes in a sandwich hybridization assay for HBV, comprising two oligonucleotides, wherein each member of the set comprises a first segment comprising a nucleotide sequence substantially complementary to a segment of E03V
nucleic acid; and a second segment comprising a nucleotide sequence substantially complementary to an oligonucleotide multimer and substantially non-complementary to said segment of HBV nucleic acid, wherein said nucleotide sequence substantially complementary to a segment of HBV nucleic acid is selected from the group consisting of:
TTGTGGGTCITrfGGGYTTTGCTGCYCCWT (SEQIDN0:6), CCTRCTCGTGTTACAGGCGGGGTiITTCIT (SEQIDN0:7), TCCATGGCTGCTAGGS1GTRC'1'GCCAAC1G (SEQIDN0:8), GCYTAYAGACCACCAAATGCCCC'TATCYTA (SEQIDN0:9), CTGTTCAAGCCTCCAAGCTGTGCCTTGGGT (SEQIDN0:10), CATGGAGARCAYMACATCAGGATTCCTAGG (SEQIDN0:11), TCCTGGYTATCGCTC,GA1'GTGTCTGCGGCGT (SEQ
ID
N0:12), GGCGCTGAATCCYGCGGACGACCCHTCTCG (SEQIDN0:13), CTTCGCTTCACCTCfGCACGTHGCATGGMG (SEQIDN0:14), - Sd - 2124928 GGTCTS1GCCAAG1GTTIGC1GACGCAACC (SEQID NO:15), CCTKCGCGGGACGTCCT1TGTYTACGTCCC (SEQID N0:16), MCCTCTGCCTAATCATCTCWIGTWCATGTC (SEQID N0:17), CGACCACGGGGCGCACCTCTCT1TACGCGG (SEQID NOslH), TGCCCAAGGTCTTACAYAAGAGGACfCTTG (SEQID N0:19), CGTCAATCTSCCRCGAGGACTGGGGACCCTG (SEQID N0:20), ATGTTGCCCGTTTGTCCTCTAM1TCCAGGA (SEQID N0:21), AT~~~T~GTfCTTCfGGAYTAYCAA (SEQID N0:22), ATCATM11'CCTCTTC11TCCTGC1GCTATGC (SHQID N0:23), CAATCACTCACCAACCTCYTGTCCTCCAAY (SEQID N0:24), GTGTCYTGGCCAAAATTCGCAGTCCCCAAC (SEQID N0:25), CTCGTGGTGGAC1TCTCTC31AT1TTCTAGG (SEQID N0:26), GACAAGAATCCTCACAATACCRCAGAGTCT (SEQID N0:27), TTTTGGGGTGGAGCCCRCAGGCT'CAGGGCR (SEQID N0:28), CACCATATTCTTGGGAACAAGARCTACAGC (SEQID N0:29), ACAC2TCCGGARACTACTGTTGTTAGACGA (SEQID N0:30), GTVTC1'fT7CGGAGTGTGGATTCGCACTCCT (SHQID N0:31), TTGGAGCWWCTGTGGAGTTAC1Y.TCRTtTf (SEQID N0:32), TTTGGGGCATGGACATYGAYCCRTATAAAG (SHQID N0:33), AAWGRTCTTTGTAYTAGGAGGCiGTAGGCA (SEQID N0:34), RGACTGGGAGGAGYTGGGGGAGGAGATTAG (SEQID N0:35), CCTTGAGGCMTACTTCAAAGACTGTICTGTT (SEQID N0:36), GTCTGTGCCTTCTCATC1GCCGGWCCG1GT (SEQID N0:37), AGCMGCTTGTTTTGCTCGCAGSMGGTCTGG (SEQID N0:38), GGCTCSTCTGCCGATCCATACTGCGGAACT (SEQID N0:39), htfKAACCTtfACCCCGTTGCTCGGCAACGG (SEQID N0:40), GTGGCTCCAGTTCMGGAACAGTAAACCCTG (SEQID N0:41), RAARCAGGCITf7CACTTTCTCGCCAAC'i'fA (SEQID N0:42) , CCTCCRCC2GCCTCYACCAATCGSCAGTCA (SEQID N0:43), ACCAATTTTCTTYTGTCTYTGGGTATACAT (SHQID N0:44).
According to yet another aspect of the invention there is provided a set of synthetic oligonucleotides useful as capture probes in a sandwich hybridization assay for HBV, comprising two oligonucleotides, wherein each member of the set comprises a first ae9ment comprising a nucleotide sequence substantially complementazy to a segment of HBV
nucleic acid; and 5e a second segment comprising a nucleotide sequence substantially complementary to an oligonucleotide bound to a solid phase and substantially non-complementary to said segment of HBV
nucleic acid, wherein said nucleotide sequence substantially complementary to a segment of HBV nucleic acid is selected from the group consisting of:
TATTCCCATCCCATC&TCCTGGGCTT'i'CGS ( SEQ ID NO ;.4 5 ) , .TATATGGATGA'IGTGGTATt'GGGGGCCAAG (SEQ ID N0:46), CGTAGGGCTTTCCCCCACZGTTT'GGCTTTC (SEQ ID N0:4~), GCTCAGTTTACTAGZGCCATZ'IGTT~UGTG (SEQ ID N0:48) , CCTATC'~GGA~GKG~C~C~Ct~TCAGYCCGTTTGTC (SEQ ID N0:49I , GTCCCCTAGA~1G~LAGAACTCCCI'C't;CCTCG ( S EQ ID NO : 5 0 ) , ACGI~GRTC'rC~ITOGCCGCGTCG~AAGA (SEQ ID NO:51) , CAATGTCGGGAATCTCAATGZ'rAGTATYCC (SEQ ID N0:52), 6ACTCATAAGGTSGGRAAC1'ITAC1CGGGCT (SEQ ID NO: 53 ) .
According to another aspect of the invention there is provided a solution sandwich hybridization assay for detecting the presence of HBV in a sample, comprising (a) contacting the sample under conditions of specific hybridization with an excess of (i) a set of synthetic oligonucleotides useful as amplifier probes in a sandwich hybridization assay for HBV
and (ii) a set of capture probe oligonucleotides wherein the capture probe oligonucleotide comprises a first segment comprising a nucleotide sequence that is substantially complementary to a segment of HBV nucleic acid and a second segment that is substantially complementary to an oligonucleotide bound to a solid phase and substantially non-complementary to said segment of HBV nucleic acid;
(b) contacting the product of step (a) under hybridizing conditions with said oligonucleotide bound to the solid phase;
5f (c) thereafter separating materials not bound to the solid phase;
(d) contacting the bound product of step (c) under hybridization conditions with a nucleic acid multimer, said multimer comprising at least one oligonucleotide unit that is substantially complementary to the second segment of the amplifier probe polynucleotide and a multiplicity of second oligonucleotide units that are substantially complementary to a labeled oligonucleotide;
(e) removing unbound multimer;
(f) contacting under hybridizing conditions the solid phase complex product of step (e) with the labeled oligonucleotide;
(g) removing unbound labeled oligonucleotide; and (h) detecting the presence of label in the solid phase complex product of step (g).
According to another aspect of the invention there is provided a solution sandwich hybridization assay for detecting the presence of HBV in a sample, comprising (a) contacting the sample under conditions of specific hybridization with an excess of (i) a set of amplifier probe oligonucleotides wherein the amplifier probe oligonucleotide comprises a first segment comprising a nucleotide sequence substantially complementary to a segment of HBV nucleic acid and a second segment comprising a nucleotide sequence substantially complementary to an oligonucleotide unit of a nucleic acid multimer and (ii) a set of synthetic oligonucleotides useful as capture probes in a sandwich hybridization assay for HBV;
(b) contacting the product of step (a) under hybridizing conditions with said oligonucleotide bound to the solid phase;
- Sg - 212492a (c) thereafter separating materials not bound to the solid phase;
(d) contacting the bound product of step (c) under hybridization conditions with a nucleic acid auiltimer. said multimer comprising at least one oligonucleotide unit that is substantially complementary to the second segment of the amplifier probe polynucleotide and a multiplicity of second oligonucleotide units that are substantially complementary to a labeled oligonucleotide;
(e) removing unbound multimer;
(f) contacting under hybridizing conditions the solid phase complex product of step (e) with the labeled oligonucleotide;
(g) removing unbound labeled oliqonucleotide;
and (h) detecting Che presence of label in the solid phase complex product of step (g).
According to another aspect of the invention there is provided a kit for the detection of HBV in a sample comprising in combination (i) a set of amplifier probe oligonucleotides wherein the amplifier probe oligonucleotide comprises a first segment comprising a nucleotide sequence substantially complementary to a segment of HHV nucleic acid and a second segment comprising a nucleotide sequence substantially complementary to an oligonucleotide unit of a nucleic acid multimer and substantially non-complementary to said segment of HBV
nucleic acid, (ii) a set of capture probe oligonucleotides wherein the capture probe oligonucleotide comprises a first segment comprising a nucleotide sequence that is substantially complementary to a segment of FD3V DNA sad a second segment that is substantially complementary to an oligonucleotide bound to a solid phase;
- Sh - z~ z49z~
(iii) a nucleic acid multimer, said multimer , comprising at least one oligonucleotide unit that is substantially complemenEary to the second segment of the amplifier probe polynucleotide and a multiplicity of aecoad oligonucleotide units that are subetantialiy complementary to a labeled oligonucleotide; and (iv) a labeled oligoaucleotide.
z~ z~9za These and other embodiments will readily occur to those of ordinary skill is view of the disclosure herein.
' , Modes for Carzvina out the Invention Definitions In defining the present invention, the following terms will be employed, and are intended to be defined as indicated below.
~SOiutioa phase nucleic acid hybridization assay" intends the assay techniques described and claimed in commonly owned U.S. Patent No: 4,868,105 sad EPA
883096976.
A "modified nucleotide' intends a nucleotide monomer that may be atably incorporated into a polyaucleotide and which has an additional functional group. Preferably, the modified nucleotide is a 5'-cytidine is which the N4-position is modified to provide a functional hydroxy group.
An 'amplifier multimer~ intends a branched polynucleotide that is capable of hybridizing simultaneously directly or indirectly to analyte nucleic acid and to a multiplicity of polyhucleotide iterations (i.e., either iterations of another multimer or iterations of a labeled probe). The branching in the multimers is effected through covalent bonds and the multimers are'composed of two types of oligonucleotide unite that are capable of hybridizing, respectively, to analyte nucleic acid or nucleic acid hybridized to analyte nucleic acid and to a multiplicity of labeled probes. The composition and preparation of such multimers are described in EPA 883096976 and W092/02526.
The term "amplifier probe" is intended as a branched or linear polynucleotide that is constructed to 7_ hav= a segment that hybridizes specifically to the ana~~ nucleic acid and iterations of a second segment that h_bridize specifically to an amplifier multimer.
The term ~captuze probe~ is intended as an oligonucleotide having a segment substantially complementary to a nucleotide sequence of the target DNA
and a segment that is substantially complementary to a nucleotide sequence of a solid-phase-immobilized probe.
~Large' as used herein to describe the comb-type branched polynucleotides of the invention intends a molecule having at least about 15 branch sites and at least about 20 iterations of the labeled probe binding sequence.
'Comb-type' as used herein to describe the structure of the branched polynucleotldea of the invention intends a polynucleotide having a linear backbone with a multiplicity of sidechaina extending from the backbone.
A "cleavable linker molecule" intends a molecule that may be atably incorporated into a polynucleotide chain and w_:ich includes a covalent bond that may be broken or cleaved by chemical treatment or physical treatment such as by irradiation.
All nucleic acid sequences disclosed herein are written in a 5' to 3' direction unless otherwise indicated. Nucleotides are designated according to the nucleotide symbols recommended by the IUPAC-IUB
Biochemical Nomenclature.
The general protocol for the solution phase sandwich hybridizations is as follows. The analyte nucleic acid is placed in a microtiter well with an excess of two single-stranded nucleic acid probe seta:
(1) a set of capture probes, each having a first binding WO 93/13t20 PCT/US92/11165 sequence substantially complementary to the analyze and a second binding sequence that is substantially complementary to nucleic acid bound to a solid support, for example, the well surface or a bead, and (2) a set of S amplifier probes (branched or linear), each having a first binding sequence that is capable of specific binding to the analyte and a second binding sequence that is capable of specific binding to a segment of the multimer. The resulting product is a three component nucleic acid complex of the two probes hybridized to the analyze by their first binding sequences. The second binding sequences of the probes regain as single-stranded segments as they are not complementary to the analyte.
This complex hybridizes to the immobilized probe on the solid surface via the second binding sequence of the capture probe. The resulting product comprises the complex bound to the solid surface via the duplex formed by the oligonucleotide bound to the solid surface sad the second binding sequence of the capture probe. Unbound materials are then removed from the surface such as by washing.
The amplification multimer is then added to the bound complex under hybridization conditions to permit the multimer to hybridize to the available second binding sequences) of the amplifier probe of the complex. The resulting complex is then separated from any unbound multimer by washing. The labeled oligonucleotide is then added under conditions which permit it to hybridize to the substantially complementary oligonucleotide units of the multimer. The resulting immobilized labeled nucleic acid complex is then washed to remove unbound labeled oligonucleotide, and read.
The analyte nucleic acids may be from a variety of sources, e.g., biological fluids or solids, and may be prepared for the hybridization analysis by a variety of WO 93/13120 ~ 1 ~ 4 9 2 8 P~/US92/11165 9_ means, e.g., proteinase K/SDS, chaotropic salts, etc.
Also, it may be of advantage to decrease the average size of the analyte nucleic acids by enzymatic, physical or chemical means, e.g., restriction enzymes, sonication, chemical degradation (e.g., metal ions), etc. The fragments may be as small as 0.1 kb, usually being at least about 0.5 kb and may be 1 kb or higher. The analyte sequence is provided in single-stranded form for analysis. Where the sequence is naturally present in single-stranded form, denaturation will not be required.
However, where the sequence may be present in double-stranded form, the sequence should be denatured.
Denaturation can be carried out by various techniques, such as alkali, generally from about 0.05 to 0.2 M
hydroxide, formamide, salts, heat, enzymes, or combinations thereof.
The first binding sequences of the capture probe and amplifier probe that are substantially complementary to the analyte sequence will each be of at least 15 nucleotides, usually at least 25 nucleotides, and not more than about 5 kb, usually not more than about 1 kb, preferably not more than about 100 nucleotides.
They will typically be approximately 30 nucleotides.
They will normally be chosen to bind to different sequences of the analyte. The first binding sequences may be selected based on a variety of considerations.
Depending upon the nature of the analyte, one may be interested in a consensus sequence, a aequeace associated with polymorphisma, a particular phenotype or genotype, a particular strain, or the like.
The number of different amplifier and capture probes used influences the sensitivity of the assay, because the more probe sequences used, the greater the signal provided by the assay system. Furthermore, the use of more probe sequences allows the use of more WO 93/13120 1 2 ~ g 2 g PCT/US92/11165 stringent hybridization conditions, thereby reducing the incidence of false positive results. Thus, the number of probes in a set will be at least one capture probe and at least one amplifier probe, more preferably two capture and two amplifier probes, and most preferably 5-100 capture probes and 5-100 amplifier probes.
Probes for HHV were designed as follows.
EPA 88309676 discloses a set of HBV probes designed by comparing the DNA sequences of the nine HHV subtypes reported in GenBank. Subsequent experimental analysis has demonstrated that these probes were complementary to the subgenomic strand (i.e. plus sense) of the incompletely double-stranded region of HHV, and thus different subsets of these probes hybridized to different viruses, since the length of the subgenomic strands varies among strains. Accordingly, the probe set has been redesigned to comprise sequences aubatantially complementary to the genomic-length strand (i.e, minus-sense) of HHV and to contain fewer spacer regions so as to include more oligonucleotides in the probe set, thereby increasing the sensitivity of the assay system.
In general, regions of greatest homology between the HBV isolates were selected as capture probes, while regions of lesser homology were selected as amplifier probes. Thus, as additional strains or isolates of HBV are made available, appropriate probes made be designed by aligning the sequence of the new strain or isolate with the nucleotide sequences used to design the probes of the present invention, and choosing regions of greatest homology for use as capture probes, with regions of lesser homology chosen ae amplifier probes. The set of presently preferred probes and their capture or amplifier overhang regions, i.e., the regions which hybridize to sequences immobilized on solid support or to an amplifier multimer, are listed in the examples.
2 1 2 4 9 2 8 P~/US92/11165 The second binding sequences of the capture probe and amplifier probe are selected to be substantially complementary, respectively, to the oligonucleotide bound to the solid surface and to a segment of the multimer and so as to not be encountered by endogenous sequences in the sample/analyte. The second binding sequence may be contiguous to the first binding sequence or be spaced therefrom by an intermediate noncomplemeatary sequence. The probes may include other noncomplementary sequences if desired.
These noncomplementary sequences must not hinder the binding of the binding sequences or cause nonspecific binding to occur.
The capture probe and amplifier probe may be prepared by oligonucleotide synthesis procedures or by cloning, preferably the former.
It will be appreciated that the binding sequences need not have perfect complementarity to provide homoduplexea. 1n many situations, heteroduplexes will suffice where fewer than about 101 of the bases are mismatches, ignoring loops of five or more nucleotides.
Accordingly, as used herein the term 'complementary"
intends exact complementarity wherein each base within the binding region corresponds exactly, and ~substantially complementary" intends 901 or greater homology.
The labeled oligonucleotide will include a sequence substantially complementary to the repeated oligonucleotide units of the multimer. The labeled oligonucleotide will include one or more molecules ("labels'), which directly or indirectly provide a detectable signal. The labels may be bound to individual members of the substantially complementary sequence or may be present as a terminal member or terminal tail having a plurality of labels. various means for providing labels bound to the oligonucleotide sequences have been reported in the literature. See, for example, Leaxy et al., proc Natl Acad Sc~ USA (1983) $Q:4045;
Renz and Kurz, Nucl. Acids Res. (1984) 1:3435;
Richardson and Gumport, Nucl. Acids Res. (1983) x:6167;
Smith et al., I~cl. Acids. Res. (1985) x:2399; Meinkoth and Wahl, Anal. Hiochem. (1984) x:267. The labels may be bound either covalently or non-covalently to the substantially complementary sequence. Labels which may be employed include radionuclides, fluorescera, chemiluminescers, dyes, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, enzyme subunita,' metal ions, and the like. Illustrative specific labels include fluoreacein, rhodamine, Texas red, phycoerythrin, umbelliferone, luminol, NADPH, a-B-galactoaidase, horseradish peroxidase, alkaline phoaphatase, etc.
The ratio of capture probe and amplifier probe to anticipated moles of analyte will each be at least stoichiometric and preferably in excess. This ratio is preferably at least about 1.5:1, and more preferably at least 2:1. It will normally be in the range of 2:1 to 106:1. Concentrations of each of the probes will generally range from about 10-5 to 10-9 M, with sample nucleic acid concentrations varying from 10-21 to 10-12 M. The hybridization steps of the assay will genezally take from about 10 minutes to 20 hours, frequently being completed in about 1 hour. Hybridization can be carried out at a mildly elevated temperature, generally in the range fran about 20°C to 80°C, more ueually from about 35°C to 70°C, particularly 65°C.
The hybridization reactions are usually done in an aqueous medium, particularly a buffered aqueous medium, which may include various additives. Additives which may be employed include low concentrations of detergent (0.01 to 1t), salts, e.g., sodium citrate (0.017 to 0.17 M), FicollT"', polyvinylpyrrolidone, carrier nucleic acids, carrier proteins, etc. Nonaqueous solvents may be added to the aqueous medium, such as dimethylformamide, dimethylaulfoxide, alcohols, and formamide. These other solvents are generally present in amounts ranging from 2 to SOt.
The stringency of the hybridization medium may be controlled by temperature, salt concentration, solvent system, sad the like. Thus, depending upon the length sad nature of the sequence of interest, the stringency will be varied.
Depending upon the nature of the label, various techniques can be employed for detecting the presence of the label. For fluorescers, a large number of different fluorometers are available. For chemilumineacera, l~nometers or films are available: With enzymes, a .
fluorescent, chemiluminescent, or colored product can be provided and determined fluorometrically, luminometrically, apectrophotometrically or visually.
The various labels which have been employed is immunoassays and the techniques applicable to immunoassays can be employed with the subject assays.
Rits for carrying out amplified nucleic acid hybridization assays according to the invention will comprise in packaged combination the following reagents:
the amplifier probe or sec of~probes; the. capture probe or set of probes; the amplifier multimer; and an appropriate labeled oligonucleotide. These reagents will typically be in separate containers in the kit. The kit may also include a denaturation reagent for denaturing the analyte, hybridization buffers, wash solutions, enzyme substrates, negative and positive controls and written instructions for carrying out the assay.
The following examples further illustrate the invention. These examples are not intended to limit the invention in any manner.
EXAMPLES
Example I
~ynrhesis of Comb tyke Branched Polvnucleotide This example illustrates the synthesis of a comb-type branched polynucleotide having 15 branch sites and sidechain extensions having three labeled probe binding sites. This polynucleotide was designed to be used in a solution phase hybridization se described in EPA 883096976.
All chemical syntheses of oligonucleotides were performed on an automatic DNA synthesizer (Applied Biosystems, Inc., (AHI) model 380 H). Phosphoramidite chemistry of the beta cyanoethyl type was used including 5'-phosphorylation which employed Phostel° reagent (AHN).
Standard AHI protocols were used except as indicated.
where it is indicated that a multiple of a cycle was used (e.g., 1.2 cycle), the multiple of the standard amount of amidite recommended by AHI waa employed in the specified cycle. Appended hereto are the programs for carrying out cycles 1.2 and 6.4 as run on the Applied Biosystema Model 380 H DNA Synthesizer.
A comb body of the following stricture was first prepared:
These noncomplementary sequences must not hinder the binding of the binding sequences or cause nonspecific binding to occur.
The capture probe and amplifier probe may be prepared by oligonucleotide synthesis procedures or by cloning, preferably the former.
It will be appreciated that the binding sequences need not have perfect complementarity to provide homoduplexea. 1n many situations, heteroduplexes will suffice where fewer than about 101 of the bases are mismatches, ignoring loops of five or more nucleotides.
Accordingly, as used herein the term 'complementary"
intends exact complementarity wherein each base within the binding region corresponds exactly, and ~substantially complementary" intends 901 or greater homology.
The labeled oligonucleotide will include a sequence substantially complementary to the repeated oligonucleotide units of the multimer. The labeled oligonucleotide will include one or more molecules ("labels'), which directly or indirectly provide a detectable signal. The labels may be bound to individual members of the substantially complementary sequence or may be present as a terminal member or terminal tail having a plurality of labels. various means for providing labels bound to the oligonucleotide sequences have been reported in the literature. See, for example, Leaxy et al., proc Natl Acad Sc~ USA (1983) $Q:4045;
Renz and Kurz, Nucl. Acids Res. (1984) 1:3435;
Richardson and Gumport, Nucl. Acids Res. (1983) x:6167;
Smith et al., I~cl. Acids. Res. (1985) x:2399; Meinkoth and Wahl, Anal. Hiochem. (1984) x:267. The labels may be bound either covalently or non-covalently to the substantially complementary sequence. Labels which may be employed include radionuclides, fluorescera, chemiluminescers, dyes, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, enzyme subunita,' metal ions, and the like. Illustrative specific labels include fluoreacein, rhodamine, Texas red, phycoerythrin, umbelliferone, luminol, NADPH, a-B-galactoaidase, horseradish peroxidase, alkaline phoaphatase, etc.
The ratio of capture probe and amplifier probe to anticipated moles of analyte will each be at least stoichiometric and preferably in excess. This ratio is preferably at least about 1.5:1, and more preferably at least 2:1. It will normally be in the range of 2:1 to 106:1. Concentrations of each of the probes will generally range from about 10-5 to 10-9 M, with sample nucleic acid concentrations varying from 10-21 to 10-12 M. The hybridization steps of the assay will genezally take from about 10 minutes to 20 hours, frequently being completed in about 1 hour. Hybridization can be carried out at a mildly elevated temperature, generally in the range fran about 20°C to 80°C, more ueually from about 35°C to 70°C, particularly 65°C.
The hybridization reactions are usually done in an aqueous medium, particularly a buffered aqueous medium, which may include various additives. Additives which may be employed include low concentrations of detergent (0.01 to 1t), salts, e.g., sodium citrate (0.017 to 0.17 M), FicollT"', polyvinylpyrrolidone, carrier nucleic acids, carrier proteins, etc. Nonaqueous solvents may be added to the aqueous medium, such as dimethylformamide, dimethylaulfoxide, alcohols, and formamide. These other solvents are generally present in amounts ranging from 2 to SOt.
The stringency of the hybridization medium may be controlled by temperature, salt concentration, solvent system, sad the like. Thus, depending upon the length sad nature of the sequence of interest, the stringency will be varied.
Depending upon the nature of the label, various techniques can be employed for detecting the presence of the label. For fluorescers, a large number of different fluorometers are available. For chemilumineacera, l~nometers or films are available: With enzymes, a .
fluorescent, chemiluminescent, or colored product can be provided and determined fluorometrically, luminometrically, apectrophotometrically or visually.
The various labels which have been employed is immunoassays and the techniques applicable to immunoassays can be employed with the subject assays.
Rits for carrying out amplified nucleic acid hybridization assays according to the invention will comprise in packaged combination the following reagents:
the amplifier probe or sec of~probes; the. capture probe or set of probes; the amplifier multimer; and an appropriate labeled oligonucleotide. These reagents will typically be in separate containers in the kit. The kit may also include a denaturation reagent for denaturing the analyte, hybridization buffers, wash solutions, enzyme substrates, negative and positive controls and written instructions for carrying out the assay.
The following examples further illustrate the invention. These examples are not intended to limit the invention in any manner.
EXAMPLES
Example I
~ynrhesis of Comb tyke Branched Polvnucleotide This example illustrates the synthesis of a comb-type branched polynucleotide having 15 branch sites and sidechain extensions having three labeled probe binding sites. This polynucleotide was designed to be used in a solution phase hybridization se described in EPA 883096976.
All chemical syntheses of oligonucleotides were performed on an automatic DNA synthesizer (Applied Biosystems, Inc., (AHI) model 380 H). Phosphoramidite chemistry of the beta cyanoethyl type was used including 5'-phosphorylation which employed Phostel° reagent (AHN).
Standard AHI protocols were used except as indicated.
where it is indicated that a multiple of a cycle was used (e.g., 1.2 cycle), the multiple of the standard amount of amidite recommended by AHI waa employed in the specified cycle. Appended hereto are the programs for carrying out cycles 1.2 and 6.4 as run on the Applied Biosystema Model 380 H DNA Synthesizer.
A comb body of the following stricture was first prepared:
3'T18(TTX')15GTTTGTGG-5' (RGTCAGTp-5')15 wherein X' is a branching_monomer, and R is a periodate cleavable linker.
The portion of the comb body through the 15 (TTX') repeats is first synthesized using 33.8 mg ,. WO 93/13120 PCT/US92/11165 a- aopropyl-derivatized thymidine controlled pore glass (CPG) (2000 A, 7.4 micromoles thymidine per gram support) with a 1.2 cycle protocol. The branching site nucleotide was of the foxacula:
O
where R2 represents O
For synthesis of the comb body (not including sidechains), the concentration of beta cyanoethylphosphoramidite monomers was 0.1 M for A, C, G
WO 93/13120 12 r~ 9 2 g PCT/US92/11165 and T, 0.15 M for the branching site monomer E, and 0.2 M
for Phostel° reagent. Detritylation was done with 3t trichloroacetic acid in methylene chloride using stepped flowthrough for the duration of the deprotection. At'the conclusion the 5' DMT was replaced with an acetyl group.
Cleavable linker R and six base aidechain extensions of the formula 3'-RGTCAGTp (SEQ ID N0:1) were synthesized at each branching monomer site as follows.
The base protecting group removal (R2 in the formula above) was performed manually while retaining the CPG
support in the same column used for synthesizing the comb body. In the case of R2 - levulinyl, a solution of 0.5 M
hydrazine hydrate in pyridine/glacial acetic acid (1:1 v/v) was introduced and kept in contact with the CPG
support for 90 min with renewal of the liquid every 15 min, followed by extensive washing with pyridine/glacial acetic acid (1:1 v/v) and then by acetonitrile. After the deprotection the cleavable linker R and nix baae sidechain extensions were added using a 6.4 cycle.
In these syntheses the concentration of phosphoramidites was 0.1 M (except 0.2 MR and Phostel°
reagent; R was 2-(4-(4-(2-Dimethoxytrityloxy)ethyl-)phenoxy 2,3-di(benzoyloxy)-butyloxy)phenyl)ethyl-2-cyanoethyl-N,N-diisopropylphosphoramiditel.
Detritylation is effected with a solution of 31< trichloroacetic acid in methylene chloride using continuous flowthrough, followed by a rinse solution of toluene/chloromethane (1:1 v/v). Branched polynucleotide chains were removed from the solid supports automatically in the 380H using the cycle ~CE
NH3.~ The ammonium hydroxide solution was collected in 4 ml screw-capped Wheaton vials and heated at 60°C for 12 hr to remove all base-protecting groups. After cooling to room temperature the solvent was removed in a Speed-Vac''N' evaporator and the residue dissolved in 100 ~1 water.
3' backbone extensions (segment A), sidechaia extensions and ligation template/linkers of the following structures were also made using the automatic ' synthesizer:
3' Backbone extension 3'-TCCGTATCC'fGGGCACAGAGGTGCp-5' (SEQ ID N0:2) Sidechain extension 3'-GATGCG(TTCATGCTGTTGGTGTAG)3-5' (SEQ ID N0:3) Ligation template for linking 3' backbone extension 3'-AAAAAAAAAAGCACCTp-5' (SEQ ID N0:4) Ligation tem-plate for link-ing sidechain extension 3'-CGCATCACTGAC-5' (SEQ ID NO: S) r The crude comb body was purified by a standard polyacrylamide gel (7% with 7 M urea and 1X TBE zunning buffer) method.
The 3' backbone extension and the sidechain~
extensions were ligated to the comb body as follows. The comb body (4 pmole/~1), 3' backbone extension (6.25 pmole/~1), sidechain extension (93.75 pmole/~1), sidechain linking template (75 pmoles/~1) and backbone linking template (5 pmole/P1) were combined in 1 mM ATP/
5 mM DTT/ 50 mM Tris-HC1, pH 8.0/ 10 mM MgCl2/ 2 mM
spermidine, with 0.5 units/W1 T4 polynucleotide kinase.
The mixture was incubated at 37°C for 2 hr, then heated in a water bath to 95°C, and then slowly cooled to below 35°C over a 1 hr period. 2 mM ATP, 10 mM DTT, 14%
polyethylene glycol, and 0.21 units/P1 T4 ligase were added, and the mixture incubated for 16-24 hr at 23°C.
The DNA was precipitated in NaCl/ethanol, resuspended in water, and subjected to a second ligation as follows.
..,~... .. The mixture was adjusted to 1 mM ATP, 5 mM DTT, 14%
polyethylene glycol, 50 mM Tris-HC1, pH 7.5, 10 mM MgCl2, 2 mM spermidine, 0.5 unita/~1 T4 polynucleotide kinase, and 0.21 units/~1 T4 ligase were added, and the mixture incubated at 23°C for 16-24 hr. Ligation products were then purified by polyacrylamide gel electrophoresis.
After ligation and purification, a portion of the product was labeled with 32P and subjected to cleavage at the site of R achieved by oxidation with aqueous NaI04 for 1 hr. The sample was then analyzed by PAGE to determine the number of sidechain extensions incorporated by quantitating the radioactive label in the bands on the gel. The product was found to have a total of 45 labeled probe binding sites.
.-.. WO 93/13120 212 ~ 9 2 ~ PCT/US92/t t 165 Hybridization Assav for HHV DNA
A "15 X 3" amplified solution phase nucleic acid sandwich hybridization assay format was employed in this example. The "15 x 3" designation derives from the fact that the format employs two multimers: (1) an amplifier probe having a first segment (A) that binds to HHV nucleic acid and a second segment (H) that hybridizes to (2) an amplifier multimer having a first segment (H~) that hybridizes to the segment (H) and fifteen iterations of a segment (C), wherein segment C hybridizes to three labeled oligonucleotides.
The amplifier and capture probe segments and their respective names used in this assay were as follows.
H6V Amplifier Probes HHV.104* (SEQ ID N0:6) HHV.94~ (SEQ ID N0:7) CCTRCTCGTGTTACAGGCGGGGTTTTTCTT
HBV.76* (SEQ ID N0:8) TCCATGGCTGCTAGGSTGTRCTGCCAACTG
HBV.87~ (SEQ ID N0:9) GCYTAYAGACCACCAAATGCCCCTATCYTA
HBV.45~ (SEQ ID NO:10) CTGTTCAAGCCTCCAAGCTGTGCCT'1GGGT
HHV.93~ (SEQ ID NO:11) CATGGAGARCAYMACATCAGGATTCCTAGG
HHV.99~ (S~Q ID N0:12) TCCTGGYTATCGCTGGATGTGTCTGCGGCGT
FIHV.78~ (SEQ ID N0:13) GGCGCTGAATCCYGCGGACGACCCHTCTCG
HSV.81~ (SEQ ID N0:14) C'ITCGCTTCACCTCTGCACGTHGCA1C~
HHV.73~070590-C (SEQ ID N0:15) GGTCTSTGCCAAGTGTTTGCTGACGCAACC
HBV.77~070590-b (SEQ ID N0:16) CCTRCGCGGGACGTCCTTTGTYTACGTCCC
HHV.D44~070590-A (SEQ ID N0:17) , '' MCCTCTGCCTAATCATCTCWT'GTWCATGTC
HHV.79~ (SEQ ID N0:1B) CGACCACGGGGCGCACCTCTCTTTACGCGG
HHV.82~ (SEQ ID N0:19) TGCCCAAGGTCTTACAYAAGAGGACTCTTG
HHV.7lt (SEQ ID N0:20) CGTCAATCTYCRCGAGGACZGGGGACCCTG
HBV.102~ (SEQ ID N0:21) ATGTTGCCCGTTTGTCCTCTAMTTCCAGGA
HHV.lOlt (SEQ ID N0:22) ATC'iTCTTRTTGGTTCTTCTGGAYTAYCAA
HBV.100~ (SEQ IDN0:23) ATCATMTTCCTCTTCATCCTGCTGCTATGC
HHV.98~ (SEQ ID N0:24) CAATCACTCACCAACCfCYTGTCCTCCAAY
HHV.97t (SEQ ID N0:25) GTGTCYTGGCCAAAATTCGCAGTCCCCAAC
HHV.96~ (SEQ ID N0:26) CTCGTGGTGGACTTCTCTCAATTTTCTAGG
HHV.95~ (SEQ ID N0:27) GACAAGAATCCTCACAATACCRCAGAGTCT
HHV.92~ (SEQ ID N0:28) TTTTGGGGTGGAGCCCRCAGGCTCAGGGCR
HBV.91~ (SEQ ID N0:29) CACCATATTCIZGGGAACppGARCTACAGC
HHV.88~ (SEQ ID N0:30) A~CTTCCGG~ACTAC'IGT'IGTTAGACGA
HHV.86t (SEQ ID N0:31) GTVTCITTYGGAGTGTGGATTCGCACTCCT
HHV.D47t (SEQ ID N0:32) ,... W093/13120 212 4 ~ 2 f3 PCT/US92/11165 HBV.D46. (SEQ ID N0:33) TTTGGGGCATGGACATYGAYCCRTATAAAG
HHV.BS (SEQ ID N0:34) AAWGRTCTTTGTAYTAGGAGGCTGTAGGCA
HHV.84 (SEQ ID N0:35) RGACTGGGAGGAGYTGGGGGAGGAGATTAG
HBV.83 (SEQ ID N0:36) CCTTGAGGCMTACTTCAAAGACTGTRTGTT
HHV.80 (SEQ ID N0:37) GTCTGTGCCTTCTCATCTGCCGGWCCGTGT
HHV.75* (SEQ ID N0:38) AGCMGCTTGTTTTGCTCGCAGSMGGTCTGG
HHV.74 (SEQ ID N0:39) GGCTCSTCTGCCGATCCATACTGCGGAACT
HBV.72* (SEQ ID N0:40) MTRAACCTiTACCCCGTTGCTCGGCAACGG
HHV.51 (SEQ ID N0:41) GTGGCTCCAGTTChGGAACAGTAAACCC2G
HHV.67 (SEQ ID N0:42) RAARCAGGCTTTYACTTTCTCGCCAACTTA
HBV.70* 062890-A (~3Q ID N0:43) CCTCCRCCTGCCTCYACCAATCGSCAGTCA
HHV.65 (SEQ ID N0:44) HHV Capture Probes HBV. 60'(SEQ ID N0:45) TATTCCCATCCCATCYTCCIGGGCZ'ITCGS
HHV. 64 (SEQ ID N0:46) TATATGGATGATGTGGTATTGGGGGCCAAG
HHV. 63'(SEQ ID N0:47) CGTAGGGCTTTCCCCCACTGTTTGGCTTTC
HHV. 62 (SEQ ID N0:48) GCTCAGTTTACTAGTGCCATTTGTTCAGTG
HHV. 61 (SEQ ID N0:49) _ CCTATGGGAGRGGGCCTCAGYCCGTTTCTC
HHV.89~ (SEQ ID NO: SO) ' GTCCCCTAGAAGAAGAACTCCCTCGCCTCG
HBV.90~ (SEQ ID NO:51) ACGMAGRTCTCMATCGCCGCGTCGCAGAAGA
HBV.D13~ (SEQ ID N0:52) . ~TCT~~TCTCJiATGTTAGTATYCC
HBV.D14~ (SEQ ID N0:53) GACTCATAAGGTSGGRAACTTTACRGGGCT
ooh ~Plifier probe contained, in addition to the sequences substantially complementary to the HBV
sequences, the following S' extension complementary to a segment of the amplifier multimer, AGGCATAGGACCCGTGTCTT (SEQ ID N0:54).
Each capture probe contained, in addition to the sequences substantially complementary to HHV DNA, the following downstream sequence complementary to DNA bound to. the solid phase (i.e, complementary to XT1~), CTTCITTGGAGAAAGTGGTG (SEQ ID NO:SS).
Microtiter plates were prepared as follows.
White Microlite 1 RemovawellT"~ strips (polystyrene microtiter plates, 96 wells/plate) were purchased from Dynatech Inc. Each well vas filled with 200 ~1 1 N HC1 and incubated at room temperature for 15-20 min. The plates were then washed 4 times with 1X PBS and the wells aspirated to remove liquid. The wells were then filled with 200 P1 1 N NaOH and incubated at room temperature for 15-20 min. The plates were again washed 4 times with 1X PHS and the wells aspirated to remove liquid.
Poly(phe-lys) was purchased from Sigma Chemicals, Inc. This polypeptide hoe a 1:1 molar ratio of phe:lys and an average m.w. of 47,900 gm/mole. It has an average length of 309 amino acids and contains 155 amines/mole. A 1 mq/ml solution of the polypeptide was mixed with 2M NaCl/1X PHS to a final concentration of 0.1 mg/m1 (pH 6.0). 100 p1 of this solution was added to each well. The plate was wrapped in plastic to prevent dzying and incubated at 30°C overnight. The plate was then washed 4 times with 1X PHS and the wells aspirated to remove liquid.
The following procedure was used to couple the oligonucleotide XT1~ to the plates. Synthesis of XT1~
was described in EPA 883096976. 20 mg diauccinimidyl suberate was dissolved in 300 ~1 dimethyl fozaamide (DMF). 26 OD260 unite of XT1~ was added to 100 ~1 coupling buffer (50 mM sodium phosphate, pH 7.8). The coupling mixture was then added to the DSS-DMF solution and stirred with a magnetic stirrer for 30 mia. An NAP-25 column was equilibrated with 10 sM sodium phosphate, pH 6.5. The coupling mixture DSS-DMF solution was added to 2 ml 10 mM sodium phosphate, pH 6.5, at 4°C.
The mixture was vortexed to mix and loaded onto the equilibrated NAP-25 column. DSS-activated XTl~ DNA was eluted from the column with 3.5 m1 10 mM sodium phosphate, pH 6.5. 5.6 OD260 units of eluted DSS-activated XT1~ DNA was added to 1500 m1 50 mM sodium phosphate, pH 7.8. 50 ~1 of this solution was added to each well and the plates were incubated overnight. The plate was then washed 4 times with 1X PHS and the wells aspirated to remove liquid.
Final stripping of plates was accomplished as follows. 200 ~L of 0.2N NaOH containing O.St (w/v) SDS
was added to each well. The plate was wrapped in plastic and incubated at 65°C far 60 min. The plate was then washed 4 times with 1X PHS and the wells aspirated to remove liquid. The stripped plate was stored with desiccant beads at 2-8°C.
g WO 93/13120 12 ~ g 2 g PCT/US92/11165 Sample preparation consisted of delivering 12.5 ~1 P-R buffer (2 mg/ml proteinase R in 10 mM Tris-HC1, pH 8.0/ 0.15 M NaCl/ 10 mM EDTA, pH 8.0/ 1t SDS/ 40 ~g/ml sonicated salmon sperm DNA) to each well.
A standard curve of HsV DNA was prepared by diluting cloned HHV, subtype adw, DNA in HHV negative human serum and delivering aliquots of dilutions corresponding co 1000, 3000, 10,000, 30,000, or 100,000 molecules to each well. Tests for cross-hybridization to heterologoua DNAs were done by adding either purified DNA or infected cells to each well. Amounts for each organism are indicated in the Table.
Plates were covered and agitated to mix samples, then incubated at 65° C to release nucleic acids.
A cocktail of the HHV-specific amplifier and capture probes listed above was added to each well (5 fmoles of each probe/well, diluted in 1 N NaOH). Plates were covered and gently agitated to mix reagents and then incubated at 65° C for 30 min.
Neutralization buffer was then added to each well (0.'7'7 M 3-(N-moipholino)propane aulfonic acid/1.895 M NaCl/0.185 sodium citrate). Plates were covered and incubated for 12-18 hr at 65° C.
After an additional 10 min at room temperature, the contents of each well were aspirated to remove all fluid, and the wells washed 2X with washing buffer (0.1t SDS/0.015 M NaCl/ 0.0015 sodium citrate).
Amplifier muitimer was then added to each well (30 fmoles/well). After covering plates and agitating to mix the contents in the wells, the plates were incubated for 30 min at 55° C.
After a further 5-10 min period at room temperature, the wells were washed as described above.
WO 93/13120 2 1 ? 4 ~ 2 $ PCT/US92/11165 Alkaline phosphatase label probe, disclosed in EP 883096976, was then added to each well (40 ~1/well of 2.5 fmoles/~1). After incubation at 55°C for 15 min, and min at room temperature, the wells were washed twice as S above and then 3X with 0.015 M NaCl/0.0015 M sodium citrate.
An enzyme-triggered dioxetane (SChaap et al., Tet. Lett. (1987) 28:1159-1162 and EPA Pub. No. 0254051), obtained from Lumiqen, Inc., was employed. 20 ~1 Lumiphos 530 (Lumigen) was added to each well. The wells were tapped lightly so that the reagent would fall to the bottom and gently swirled to distribute the reagent evenly over the bottom. The wells were covered and incubated at 37°C for 40 min.
Plates were then read on a Dynatech ML 1000 luminometer. Output was given as the full integral of the light produced during the reaction.
Results from an exclusivity study of the HHV
probes is shown in the Table below. Results for each standard sample are expressed as the difference between the mean of the negative control plus two standard deviations and the mean of the sample minus two standard deviations (delta). If delta is greater than zero, the sample is considered positive. These results indicate the ability of these probe sets to distinguish HHV DNA
from heterologous organisms and a sensitivity of about 1000-3000 H8V molecules.
Table Sample Amount Delta HHV 1 X 105 25.99 HHV 3 X 104 6.51 HBV 1 X 104 3.00 HHV 3 X 103 0.93 HBV 1 X 103 -0.20 Control __ H~ ~ 8 X i05 -0.39 ~1 3 .3X 106 -0.48 HTLV-II2 1 X 105 -0.07 HT'LV-I2 1 X 105 -0.23 HIV 1 X 107 -0.31 pBR325 1 X 107 -0.27 Streptococcus sanguis 1 X 107 -0.31 Streptococcus pyogenes 1 X 107 -0.36 Streptococcus pneumoniae 1 X 107 -0.38 Streptococcus fecalis 1 X 107 -0.28 Streptococcus agalactiae 1 X 107 -0.26 Streptococcus epidermidis 1 X 107 -0.31 Staphylococcus aureus 1 X 107 -0.34 Serratia marceacena 1 X 107 -0.30 Pseudomonas aeruginosa 1 X 107 -0.23 Proteua mir3bilis 1 X 107 -0.43 Peptostreptococcus 1 X 107 -0,46 anerobius Lactobacillus acidophilus 1 X 107 -0.33 Rlebaiella pneumoniae 1 X 107 -0.12 Haemophilus influenza 1 X 107 -0.34 Escherichia coli 1 X 107 -0.44 Enterobacter aerogenea 1 X 107 -0.23 Mycobacterium leprae 1 X 107 -0.18 1 denotes pfu in infected cells 2 denotes proviral copies Modifications of the above-described modes for carrying out the invention that are obvious to those of skill in biochemiatzy, nucleic acid hybridization assays, and related fields are intended to be within the scope of the following claims.
WO 93/13120 ~ PCT/US92/11165 _27_ SEQUENCE. LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Irvine, Bruce D.
Rolberg, Janice A.
Running, Joyce A.
Urdea, Michael S.
(ii) TITLE OF INVENTION: HBV PROBES FOR USE IN SOLUTION
PHASE SANDWICH HYBRIDIZATION ASSAYS
(iii) NUMBER OF SEQUENCES: 55 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Morrison ~ Foerster (H) STREET: 755 Page Mill Road (C) CITY: Palo Alto (D) STATE: California (E) COUyTRY: USA
(F) ZIP: 94304-1018 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (H) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: 07/813,586 (H) FILING DATE: 23-DEC-1991 (C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Thomas E. Ciotti (H) REGISTRATION NUMBER: 21,013 (C) REPERfiNCE/DOCRET NUMBER: 22300-20234.00 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 415-813-5600 (H) TELEFAX: 415-494-0792 (C) TELEX: 706141 (2) INFORMATZON FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ZD NO:1:
TGACIGFL
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs (B) TYPE: nucleic acid (C) STRANDEDNBSS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
GATGTGGTTG TCGTACTTGA TGTGG!'1GTC GTACIZGATG TGGTTGTCGT AC11GCGTAG 60 (2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs (H) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
(2) INFORMATION FOR SEQ ID N0:5:
(i> SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 base pairs WO 93/13120 ~ PCf/US92/11165 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear 5 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: S:
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs 10 (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
TTGTG('aGTCT 2TIGGGYTIT GC'fGCYCCWT 30 (2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid 20 (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
CCrICCTCGT'G TTACJVGGCGG GGTSTITCI1' 30 (2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUBNCB CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single 30 (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
TCCAT'GGCTG CTAGGSI~,lR CTGCCAAClG 30 35 (2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
(2) INFORMATION FOR SEQ ID N0:10:
10 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:10:
CTGTTCJ1AGC CTCCAAGCfG 1GCCT~GGGT' 30 (2) INFORMATION FOR SEQ ID N0:11:
(i) SEQUENCE CHARACTERISTICS:
20 (A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear 25 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
(2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCB CHARACTERISTICS:
(A) LENGTH: 31 base pairs 30 (H) 'TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
WO 93/13120 1 2 ~ 9 2 g PCT/US92/11165 (2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:
GGCGC'TGAAT CCYGCGGACG ACCCBTCTCG 30 (2) INFORMATION FOR SEQ ID N0:14:
(i1 SEQUENCfi CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:
CT1'CGCTTCA CCTC1~GCACG TFIGCA1'GQ~Ti 30 (2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCB CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:15: ' GG1'CfST'GCC AAGTG'ITIGC TGACGCAACC 30 (2) INFORMATION FOR SEQ ID N0:16:
(i1 SEQUENCE CHARACTERISTICS:
(A) LfiNGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDHDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCfi DESCRIPTION: SEQ ID N0:16:
CC17CCGCGGG ACGTCCITiG TYTACGTCCC 30 (2) INFORMATION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:
MCCTCTGCCT AATCATCTC4I TGTSiCATGTC 30 (2) INFORMATION FOR SEQ ID N0:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:18:
(2) INFORMATION FOR SEQ ID N0:19:
(i) SEQUENCB CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ZD N0:19:
~'CCCAAGGT C1TACAYAAG 71GGACfC'ITG 30 (2) INFORMATION FOR SEQ ID N0:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs -(H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear WO 93/13120 1 ~ ~ 9 2 g PCT/US92/11165 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:20:
CGTCAATCTY CKCGAGGALT GGGGACCCfG
~ 37 5 (2) INFORMATION FOR SEQ ID N0:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:21:
ATGTTGCCCG TTTGTCC'f'CT AM! I'CC~,C,p (2) INFORMATION FOR SEQ ID N0:22:
15 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (Dl TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:
ATCTTCTTRT TGGTTCTTCT GGAYTAYCAA
(2) INFORMATION FOR SEQ ID N0:23:
(i) SEQUENCE CHARACTERISTICS:
25 (A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear 3p (xi) SEQUENCE DESCRIPTION: SEQ ID N0:23:
(2) INFORMATION FOR SEQ ID N0:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid WO 93/13120 2 1 2 4 9 2 8 P~/US92/11165 (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:24:
CAATCACTCA CCAACCfCYT GTCCTCCAAY 30 (2) INFORMATION FOR SEQ ID N0:25:
(i1 SEQUENCfi CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid to (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:25:
GTGTCYT'GGC CAAAATTCGC AGT'CCCCWC 30 (2) INFORMATION FOR SEQ ID N0:26:
(i) SEQUENCB CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNBSS: single (D) TOPOLOGY: linear (xi) SEQUENCB DfiSCRIPTION: S8Q ID N0:26:
CTCGTGGTGG ACTCCTCrCA ATTTTCTAGG 30 (2) INFORMATION FOR SEQ ID N0:27:
(i) SEQUENCB CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUBNCE DESCRIPTION: SEQ ID N0:27:
GACAAGAATC CTCACAATAC CRCAGArTCT 30 (2)INFORMATION FOR SEQ ID N0:28:
WO 93/13120 ~ 12 ~ 9 2 8 PCT/US92/I 1 t65 (i> SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:28:
(2) INFORMATION FOR SEQ ID N0:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single ' (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:29:
CACCATATTC TTGGGAAC)1A GAKCTACAGC 30 (2) INFORMATION FOR SEQ ID N0:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDSDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:30:
ACACTTCCGG ARAC1'ACfGT' TGTTAGACGA 30 (2) INFORMATION FOR SEQ ID N0:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:31:
GTVI'CITrYG GAGIGTGGAT TCGCACTCCS 30 WO 93/13120 ~ 12 ~ 9 2 8 PCT/US92/11165 (2) INFQRMATION FOR SEQ ID N0:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:32:
T1'GGAGCWWC TGTG«AG1TA CTCTCKTTrT 30 (2) INFORMATION FOR SEQ ID N0:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQU~NCB DESCRIPTION: SEQ ID N0:33:
(2) INFORMATION FOR SEQ ID N0:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:34:
AAWGRTCZTr GTAYTAGGAG GClGTAGGC~1 30 (2) INFORMATION FOR SEQ ID N0:35:
(i1 SEQUENCB CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ~12~928 WO 93/13120 PCf/US92/11165 _37_ (xi) SEQUENCE DESCRIPTION: SEQ ID N0:35:
RGAC'IGGC''AG GAGYTGGC,GG AGGAGATTAG
(2) INFORMATION FOR SEQ ID N0:36:
5 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear 10 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:36:
CCTTGAGrCM TACTT'CJIpT~G A~,T~.~
(2) INFORMATION FOR SEQ ID N0:37: w (i) SEQUENCE CHARACTERISTICS:
15 (A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCB DESCRIPTION: SEQ ID N0:37:
cTCrcTCCCr TcrcaTCrcc cccwcccn;T
(2) INFORMATION FOR SEQ ID N0:3B:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid 25 (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCB DESCRIPTION: SEQ ID N0:38:
3 0 ~ T~~G csteccTCrcc (2) INFORMATION FOR SEQ ID N0:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRAND&DNfiSS: single 35 (D) TOPOLOGY: linear WO 93/13120 PCr/US92/11165 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:39:
GGCTCSTCfG CCGATCCATA CTGCGGAACT ~ 30.
(2) INFORMATION FOR SEQ ID N0:40:
(I) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:40:
(2) INFORMATION FOR SEQ ID N0:41:
(I) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SSQ ID N0:41:
GTGGCTCCAG TTC29'.,GAACA GTAAACCCTG 30 (2) INFORMATION FOR SEQ ID N0:42:
(I) SEQUENCB CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:42:
KAARCAGGCT TTYAC1TIY.T CGCCAACTTA 30 (2) INFORMATION FOR SEQ ID N0:43:
(I) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs 3S (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:43:
CCTCCKCCTG CCtCYACCAA TCGSCAGTCA 30 (2) INFORMATION FOR SEQ ID N0:44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:44:
ACCAATTTTC T1'YTGIrTYT GGGTATACAT 30 (2) INFORMATION FOR SEQ ID N0:45:
(i) SEQUfiNCB CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:45:
TATTCCCATC CCATCRTCCT GGGC1'P1'CGS 30 (2) INFORMATION FOR SEQ ID N0:46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:46:
(2)INFORMATION FOR SEQ ID N0:47:
WO 93/13120 ~ ~ ~ ~ ~ ~ ~ PCT/US92/t 1165 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:47:
CGTAGGGCIT TCCCCCACfG TTTGGCI1TC 30 (2) INFORMATION FOR SEQ ID N0:48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single -(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:48:
GCTCAGTTTA CfAGIGCCAT TTGTTCAGTG 30 (2) INFORMATION FOR SEQ ID N0:49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:49:
CCTATGGGAG KGGGCCTCAG YCCGTTI'CTC 30 (2) INFORMATION FOR SEQ ID NO:50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:50:
GTCCCCTAGA AGAAGAACTC CCTCGCCfCG 30 ", WO 93/13120 ~ 1 2 ~ g 2 g PCT/US92/11165 (2) INFORMATION FOR SEQ ID N0:51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:51:
ACGMAGRTCT CMATCGCCGC C1'CGCAGAAG A 31 (2) INFORMATION FOR SEQ ID N0:52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:52:
(2) INFORMATION FOR SEQ ID N0:53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single, (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:53:
GACTCATAAG GTSGGRAACT TTACKGGGCZ' 30 (2) INFORMATION FOR SEQ ID N0:54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear WO 93/13120 12 4 ~ 2 g PCT/US92/1116s (xi) SEQUENCB DESCRIPTION: SEQ ID N0:54:
AGGCATAGGA CCCG1'GTCZT 20 (2) INFORMATION FOR SEQ ID NO:55:
(i1 SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear 10 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:55:
crrcrrrcca caraacTCCrc ?o WO 93/13120 ? 1 2 L~ 9 2 ~ PCT/US92/11165 FIL°_ NP11E LAST ACCE66 OAT~ ~..~EFTEC cILE NAHE ~9T ACCES- PATE
C2cRTrC
FILE TY°E~ 9YNTHE6I5 CYCL
%SC-~ 08 Z7. 199108 27r.1991 o.a%9-S 08 ZT, 199i08 '_'.laal 6. "
I.Z%D-n08 27. 199108 27,1991 1.2%-0 08 27. 199108 Z7.1991 aseeef30t 07, 19900t 07,1990 eeaf3 0t 07, 19900t 07,1990 lOeeeP321 07, 199001 07,1998 naaf3 0i 07, 199001 07,1990 l0haaf30t 07, 199001 27.,1990 rnsaf3 01 07, 1990Bt 07,1990 t0rnaaf3-01 87, 199001 07,I99B saeef3 0I 07, 199001 07,1998 cef3 01 07, 199001 07,1998 t0eaf3 01 07, 199001 07,1990 l0hof3 81 07, 19900t 07,i990 rnsf3 01 07. 19900t 07.1990 t0rnaf301 07, 199001 07,1990 aaeaaft 01 07, 19980f 07,1990 ceafl 0I 07, 19900I 07,1990 IOeeafl 01 07, 199001 07,1990 noaft 0t 07, 199001 07,1990 t0hoafl 0t 07, 199001 07,1990 rneafl 01 07. 199001 07,1990 l0rnaafl 01 87, 199001 07,1990 asceft 01 07, 199001 07,1990 esfl 01 A7, 199001 07,1990 l0ceft 0t 07, 199001 07,1990 l0hofl 0t 07, 19900t 07.1990 rnalt 01 07, 1990AI 07,1990 l0rnefl 01 07, 199001 07,1990 FILE TYPE: BOTTLE CHAH6E PROCEDURE
be 18 07 01, 1986 07 01. 1986 be 17 07 01, 1986 07 01, 1986 tie i6 07 01, 1986 07 0t, 1986 be 15 07 8t, 1986 87 01, 1986 be to 07 01, 1986 07 01, 1986 be 13 07 81. 1986 87 01, 1986 be IZ 07 0t, 198fi 07 81, 1986 be 11 07 81, 1986 07 01, 1986 be 10 07 01, 1986 07 01, 1986 be 9 07 01, 1986 07 01, 1966 he 8a 07 01. 1966 07 01. 1986 be 7 07 01. 1986 07 01, 1986 be a 07 01, 1986 07 01, 1986 be 5 87 01, 1986 07 01., 1986 tie a 07 01, 1986 07 0I, 1986 tic 3 87 01, 1986 07 0t, 1986 oc 2 07 01, 1986 07 0t~, 1986 be I 07 0I, 1986 07 Bt, 1986 FILE TYPE:. ENO PROCEDURE
CAP-PRIM '08 2T. 1991 08 27. 1991 CE NH3 88 Z7, 1991 88 27. 1991 deoree 10 88, 1990 18 88. 1990 deoreel0 10 08, 1990 t0 08, 1990 deorho 18 08, 1990 I0 08, 1998 deorhol0 10 0B, 1998 18 08, 1990 deorna t0 08, I99A 10 08. 1998 deprnal0 10 08. 1990 10 08, 1990 FILE TYPE: 8E6IH PROCEDURE .
STD PR~° 88 27, 1991 88 27. 1991 onos803 87 81, 1966 07 01, 1986 FILE TYPE: SHUT-0O~H PROCEDURE
clean083 87 01, 1986 A7 81, 1986 FILE TYPE: DNA SEOUEHCE9 I6%-. 88 Z7, 1991 08 27. 1991 _ IS%-I 08 27, 1991 0B 2'. 1991 WO 93/13120 2 1 2 4 9 2 8 P~/US92/11165 4 ~I
STEP FUNCTION STEP STEP ACTIVE FOR 8R9E9 SrlFc N M . 3 LARNE T-.~F. R- 6 r 1 10 i19 To Ueate 3 Yes Yes Yes Yea Yea Yea Yes Yea ? 3 i16 To Column10 Yea Yes Yea Yea Yes Yea Yes Yea 3 Z Reverse Flusn~ Yea Yes Yes Yes Yea Yes Yes Yea s I Bloek Fluan 3 Yes Yes Yea Yea Yes Yes Yes Yea ~ Aovenee FC t Yea Yea Yea Yea Yes Yea Yes o' 29 . 3 Yea Yes ~ it5 Phoa Preo 1 Yea Yea Yea Yea Yea Yes Yea 6rouo I On Yea Yea Yea Yes Yea Yaa Yes Yea 8 98 TET To Coluwn1A Yea Yes Yea Yes Yes Yea Yes Yaa 9 19 6aTET To Col 6 Yea Yes Yes Yea Yes Yes Yes I Yea 9A TET To Columns Yes Yes Yes Yea Yea Yea Yes 11 -a6 6rouo 1 Off I Yes Yea Yes Yea Yea Yea Yes Yea Yea IZ .a7 6rouo 2 On 1 Yea Yea Yes Yes Yes Yes Yes Yea 13 90 TcT To Coluwn10 Yea Yea Yea Yea Yea Yaa Yea Yea is 28 9TET To Col 8 Yea Yea Yea Yes Yes Yea Yea 2 Yea 98 TET To Column' Yea Yea Yes Yea Yea Yea Yea Yea I6 -t8 6rouo 2 Off 1 Yea Yes Yes Yea Yea Yea Yea 17 it9 6rouo 3 On I Yea Yea Yes Yea Yea Yea Yea Yea Yea I6 98 TET To Coluwn18 Yea Yes Yaa Yea Yea Yea Yea 19 21 8TET To Col 8 Yea Yea 28 98 3 t Yes Yea Yea Yea Yea Yea Ye TET To Coluwn Yea Yea Yees Yea Yea Ysa Yea Yea a 21 -58 6rouo 3 Off 1 Yes Yea Yaa Yas Yea Yea Yes ZZ t Uett IS Yea Yes 23 ~S 6rouo I On 1 Yes Yea Yea Yaa Yes Yea Yea Zs 90 Tc~ To Coluwn18 Yea Yea Z5 i9 BTET To Col 6 Yes Yes Yea Yea Yes Yea Yes 26 90 1 4 Yna Yea Z7 -t6 TET To Column1 Yea Yes Yea Yea Yea Yea Yea 26 ~7 6rouo 1 Ott 1 Yes Yea 29 90 6rouo 2 On 10 Yea Yea Yea Yea Yes Yea Yea 38 ~ 7ET To Coluwn8 Yea Yes 31 Z8 BtTET To Col t Yea Yes Yea Yea Yea Yes Yes 32 9A Z I Yaa Yes 33 -t8~ TET To Column1 Yea Yes Yea Yea Yea Yea Yea 34 it9 Group 2 Otf 18 Tea Yes 35 9A 6rouo 3 On 8 Yes Yea Yea Yea Yea Yes Yea 36 21 TE7 To Coluwn1 t Yea Yea 37 98 BfTET To Cal t Yea Yea Yea Yas Yea Yea Yea 38 -58 3 ' 3B Yea Yes 39 t TET To Coluwn1 Yes Yes Yea Yaa Yea Yes Yea t8 X45 6rouo 3 Otf 10 Yea Yes t1 98 fait B Yea Yea Yea Yea Yea Yea Yea tZ 19 6rouo I On t Yea Yer s3 9A TET To Coluwnt Yaa Yes Yea Ysa Yea Yea Yea -tfi BtTET To Col Yea 1 Yas Yes Yaa Yea Yea Yea TET To Coluwn Yes 6rouo 1 Otf Yea Yea Yes Yaa Yea~YSa Yes Yea Yea Yea Yes Yea Yea Yes Yaa Yea Yea Yea Yea Yes Yee Yva Yea YeaYaa Yes Yes Yea Yas Yes Yea Yaa Yea Yea Yu Yea Yea Yes Yea Yea Yea Yas Yea Yes Yea Yes Yea Yea Yes Yea Yea Yea Yea Yea Yea Yes Yea Yea Yea Yea Yea Yea Yea Yea Yea Yes Yea Yes Yes Yea <Conttnueo next OaOe.>
WO 93/13120 ~ 12 4 9 2 8 PC1'/US92/11165 STEP FUNCTION STEP STEP ACTIVE FOR HR9E9 SAF=
NUhBEFt NH11~ T M 9 ~ ~ ' -' S--n al X67 or9Up Z ~n t Yea Yea Yea Yea Yea Y.a Yea ~~a 6~ 90 TET To Column 10 Yea Yes Yea Yea Yea Yea Yes tes a6 20 B~TET To Col : 9 Yes Yes Yes Yea Yes Yea Yea taa a7 90 TET To CoLuwn a Yes Yea Yes Yes Yea Yes Yea iaa a8 -a8 Group 2 Off 1 Yes Yea Yea Yea Yes Yea Yea Y-_a d9 ~a9 6rouo 3 On t Yes Yea Yea Yea Yna Yea Yea Yea 90 TET To Coluwn 10 Yea Yea Yea Yea Yea Yea Yea res 51 . 21 6~TE7 To Col 3 ' B Yea Yea Yea Yea Yea Yea Yaa Yes 52 90 T-ET To Coluwn a Yaa Yea Yes Yea Yea Yea Yea Yea 53 -50 6rouo 3 Off t Yea Yea Yea Yes Yea Yes Yas Yes 5a a yslt 30 Yes Yea Yna Yea Yes Yes Yea Yea ~a5 orouo 1 On 1 Yea Yea Yes Yea Yea Yea Yea Yea S6 90 TET To Coluwn 10 Yea Yea Yea Yes Yea Yea Yea Yes 57 I9 HrTET to Col t 8 Yea Yes Yes Yea Yea Yea Yes Yes 58 90 TET To Coluwn a Yes Yes Yea Yea Yea Yes Yes Yaa 59 -a6 Group I Off I Yea Yaa Yea Yea Yea Yea Yes Yes ~a7 Group 2 On I Yea Yea Yea Yea Yea YeaYea Yea 6I 90 Tc7 To Column 10 Yes Yea Yaa Yes Yea Yea Yea Yea 6Z 20 HtTET To Col 2 8 Vas Yaa Yea Yar Yea Yea Yes Yea 63 9A TET To Coluwn 4 Yes Yes Yea Yea Yea Yes Yea Yea 6a -a8 Group 2 Off 1 Yea Yas Yea Yea Yaa Yaa Yea Yea ~a9 Group 3 On I Yea Yes Yea Yaa Yaa Yas Yea Yes 66 90 TET To Coluwn 1H Yea Yea Yes Yea Yea Yea Yes Yes 67 Z1 H~TET To Col 3 8 Yea Yes Yaa Yes Yaa Yes Yea Yea 66 9A TET To Colwn a Yea Yea Yea Yea Yea Yea Yea Yea 69 -58 Group 3 Off 1 Yea Yea Yea Yes Yas Yea Ysa YGs a ualt 30 Yea Yea Yea Yea Yea Yea Yea Yes 7t ~a5 Group I On I Ysa Yes Yea Yea Yea Yea Yea rea 72 90 TET To Coluwn 10 Yes Yea Yaa Yea Yea Yea Yea Yea 73 19 BtTET To Col 1 8 Yea Yea Yea Yea Yes Yea Yea Yea 7a 9A TET To Colwn a Yaa Yaa Yea Yea Yea Yes Yes Yes -a6 Group 1 Off I Yes Yea Yea Yaa Yea Yea Yes Yes 76 '~a7 firouo 2 On 1 Yss Ysa Yes Ysa Yea Yaa Yaa Yes 77 9i TET To Colwn 10 Yea Yaa Yas Yes Yes Yea Yea . Yes 7H 2~ B~TET To Col 2 8 Yas Yes Yea Yaa Yea Yaa Yea Yea 79 9i TET To Colwn a Vas Yea Yes Ysa Yea Yaa Yea Yea HA -a8 Group Z Off I Yes Yea Yea Yea Yes Yes Yes Yes Bt ~a9 6reup 3 On I Yea Yea Yea Yea Yes Yaa Yea Yes H2 9H TET To Coluwn ~ 10 Yss Yea Yes Yea Yea Yea Yes Yea 63 21 HaTET To Col 3 H Yea Yea Yee Yea Yaa Yea Yes Yea H4 90 TET Ta Colwn a Yea Yea Yea Yea Yea Yea Yea Yes HS -5B Group 3 Off I Yea Yea Yaa Yea Yea Yas Yes Yea H6 a yell 3H Yes Yes Yas Yas Yea Yes Yaa Yea H7 ia5 Group I On 1 Yaa Yaa Yaa Yea Yaa Yea Yns Yea 88 9H TET 7o Colwn I0 Yes Yes Yaa Vas Yea Yea Yes Yea_ (Coneinued neat ps0e->
WO 93/13120 ~ 2 ~ 9 2 8 PCT/US92/1 I 165 9TE? FUNCTION 9TE. gTE? ACTIVE FOfY 8H9E9 SAFc N h x NtIwE TIME 9 r r E '~ c-.~~
g9 19 GiET To Col _ 0 Yas Yes Yea Yes Yes Yes Yes I Yes 9p 9B iET To Column .t Yes Yes Yea Yes Yea Yes Yes Yes 91 -La 6rou0 1 Off I Yea Yes Yes Yea Yea Yes Y-_s Yes g2 +a7 Grouo 2 On t Yea Yea Yes Yes Tea Yea Yes Yes 93 90 TET To Coluwn 1B Yes Yea Yes Yes Yes Yea Yes Yes 9a Z8 B+TET To CoL 8 Yes Yea Yes Yea Yea Yes Yes 2 Yes 95 98 TET To Coluwn a Yes Yea Yes Yea Yea Yea Yes ~ Yea 96 ~ 6rou0 2 Oft 1 Yea Yea Yea Yea Yes Yes Yes -a8 Yes g7 +a9 Group 3 On I Yea Yes Yea Yas Yaa Yes Yes Yes 9A 90 TcT To Coluwn I8 Yea Yes Yes Yea Yes Yes Yes Yes 99 2i B+TET To Co1 B Yea Yea Yea Yes Yes Yea Yea 3 Yes 180 98 T'cT To Coluwna Yaa Yes Yes Yaa Yes Yea Yes Yea I81 -tea Group 3 Oft I Yea Yes Yea Yes Yes Yea Yea Yea 182 a Wait 30 Yes Yea Yea Yea Yea Yes Yes Yea 103 a6 6rouo I On 1 Yna Yea Yes Yea Yea Yea Yes Yea 10a 98 TET To Coluwn 10 Yea Yes Yea Yea Yes Yes Yes Yea 105 19 B+TET To Col 8 Yea Yea Yaa Yea Yaa Yea Yes I Yea 186 98 TcT To Coluwn Yea Yea Yea Yea Yes Yes Yea Yea 107 -a6 Group 1 Otf I Yea Yea Yaa Yea Yaa Yea Yes Yea 188 +a7 Group 2 On I Yaa Yea Yaa Yea Yas Yea Yea Yea 189 9A TET To Coluwn 18 Ysa Yaa Yaa Yaa Yaa Yes Yea Yaa II0 28 8+TET To Col 8 Yas Yaa Yea Yea Yaa Yas Yea 2 Yaa 111 90 TET To Coluwn t Yes Yes Yea Yes Yea Yea Yea Yea 112 -t8 Group 2 Off 1 Yes Yes Yea Yea Yea Yes Yes Yea 113 +a9 Group 3 On 1 Yea Yaa Yas Yea Yes Yea Yes Yes 4 98 wn 18 Yaa Yea Yea Ysa Yea Yes Yea TET To Col Yea 11 21 u A Yea Yas Yea Yea Yaa Yea Yea I15 6+TET To Col Yea 116 98 TET To Coluwn a Yes Yea Yaa Yes Yea Ysa Yea Yea 117 -Sa Group 3 Oft I Vas Yea Yea Yea Yea Ysa Yea Yea 118 a hut 38 Ysa Yaa Yea Yea Yaa Yea Yes Yes 1i9 +a5 Group I On 1 Yea Yes Yea Yaa Yes Yea Yes Yes 120 90 TET To Coluwn IA Yaa Yes Yea Yaa Yea Yes Yes Yea I21 ' B+TET To Col A Yes Yas Yes Yaa Yaa Yes Yea 19 1 Yaa 122 9i TET To Cpluwn L Yea Yes Yaa Yas Yea Yea Yes Yes 123 -I6 Group I Off I Yea Yes Yas Yaa Yes Yea Yea Yaa I2a +a7 6rouo 2 On 1 Yes Yas Yea Yaa Yes Yea Yes Yaa 125 9A TET~ To ColuwnIA Yea Yea Yea Yes Yea Yes Yea Yea I26 2i 8+TET To Col 8 Vas Yaa Yea Yea Yaa Yes Yes 2 Yaa 127 98 TcT To Coluwn ~ a Yea Yea Yea Yea Yea Yes Yea Yea 128 -a8 Group 2 Off 1 Yaa Yaa Yea Yu Yas Yaa Yes Yea 129 a9 Group 3 On I Yea Yae Yee Yae Yea Yea Yes Yea 138 9A TET To Caluwn 18 Yea Yea Yaa Yas Yea Yea Yes Yas 131 2I B+TT To Col 8 Yea Yes Yes Yea Yes Yea Yea 3 Yes 132 98 TET To Coluwn 4 Yaa Yes Yes Yes Yaa Yea Yns Yes 133 -58 6rouo 3 Off I Yes Yaa Yea Yea Yaa Yes Yes Yes _ (Conttnueo next oepa.l .... W093/13120 ~ pCT/US92/Itl6;
STE? FUNCTION STEP STEP HCTIVE
iAFc FAR
BHSEi N h s NllnE TIhE R 5 y > > ' iT~
i3d 1 :lest ~0 Yes YeaYeaYesYesYeaYes Yes 135 id 318 To Uaate i Yes YesYesYesYesYeaYts Yea 136 . Reverse Fluah~ Yes YeaYeaYeaYesYeaYes Yes 137 1 8lotk Flush d Yea YesYeaYeaYeaYeaYes Yea 138 31 SIS To usate 3 Yea YesYesYeaYeaYeaYes Yea 139 13 f15 To Column22 Yea YeaYeaYesYesYesYea Yes 1a0 10 f18 To 4laateS Yea YeaYeaYeaYeaYeaYea Yea 1t1 d ~ett 30 Yea YeaYeaYaaYesYeaYea Yea It2 2 Reverse Fluah6 Yea YeaYeaYeaYeaYeaYea Vas 1t3 I Block Fluah Yes YeaYesYeaYesYeaYea Yea tat ? fIB To Column10 Yes YeaYeaYeaYeaYeaYea Yea 115 3d FLuah to Waate~ Yea YeaYeaYesYesYesYea Yea I46 3 318 To Column10 Yea YesYeaYeaYesYesYea Yea 1a7 . Reverse Fluan5 Yea YeaYesYeaYeaYeaYea Yes It8 9 a18 To Columnf0 Yea YeaYesYeaYeaYeaYes Yes It9 2 Reverse FlusnS Yea YeaYeaYeaYeaYesYea Yes I50 9 f18 To Column10 Yea YeaYesYeaYesYesYea Yea 151 2 Reverse Fluah5 Yea YeaYesYeaYeaYesYes Yea 152 1 Hloek flush t Yes YesYesYeaYasYesYes Yes 153 33 Cyela Entry t Yas YeaYesYssYesYesYea Yen 154 5 4laate-Port I Yas YaaYsaYeaYeaYeaYea Yea 155 37 Ralay 3 PulseI Yaa YeaYeaYesYeaYeaYsa Yes 156 82 ilt To ~aate 3 Yea YaaYesYeaYeaYesYea Yea 157 30 3l7 To haste 3 Yea YeaYeaYeaYeaYeaYea Yea I58 10 f18 To yaste S Yea YeaYesYeaYeaYeaYaa Yea 159 9 t18 To Column20 Yea YeaYeaYesYeaYesYea Yes 160 II :l7 To Column60 Yea YeaYesYeaYesYeaYes No 161 1d fIL 7o Column20 Yea YesYeaYeaYeaYeaYea No 162 2 Reerse Fluah 7 Yes YesYeaYesYesYeaYea No 163 11 t17 To ColumnIS Yea YeaYeaYeaYsaYeaYea No I6t 3t Fluah to haste-5 Yes YaaYeaYeaYesYeaYea No I65 11 ill To ColumnIS Yes YeaYeaYeaYeaYesYes No I66 ' Rweraa Flush 5 Yea YeaYeaYeaYeaYsaYes Ho l67 It ttt 7o Colwn 20 ~ YssYasYsaYeaYesYea No Yes 168 .3t Fluah to haste10 Yes YeaYasYesYeaYesYea No 169 7 4laate-BottlsI Yea YeaYesYsaYeaYeaYea Yes 170 9 f18 To Column10 Yea Yea Yes YsaYea Yea Yea Yea 171 2 Reveraa Flush5 Yes Yas Yea YasYes Yes Yes Vas 172 9 fib To Calunn~ 10 Yes Yes Yea Yes Yss Yea Yea Yea 173 Z Reveraa FlushS Yea Yea Yea Yea Yea Yes Yea Yaa 17t 9 i18 To Column10 Yes Yea Yea Yaa Yae Yas Yea Yea 175 2 Revsrae Fluah5 Yes Yes Yes Yes Yea Yea Yea Yes ' i76 1 0loek Fluah 3 Yea Yea Yes Yea Yea Yea Yea Yes WO 93/13120 ~ PCT/US92/11165 STEP FUNCTION STEP STEP RCTIV FOR BR9E9 SRF=
NUN9ER 3 TINE A ~ r T 5 'T 5~F' NRNF
I 10 S18 To ~aate 3 Yea Yes Yes Yes Yes Yea Vea Yea 2 9 a18 To Column10 Yes Yes Yea Yes Yes Yes Yaa Yea S 2 Reverse Fluan~ Yes Yea Yes Yea Yes Yea Y-_a a I Block Flush 3 Yea Yea 5 Advenee FC I Yes Yea Yea Yea Yes Yea Yes 6 ~ Phos Prep 3 Yea Yes ? 29 Group 1 On 1 Yea Yes Yea Yaa Yaa Yaa Yea 8 +a5 TET To ColumnI0 Yea Yea 9 90 B+TET To Col 8 Yea Yea Yea Yes Yes Yea Yes 19 1 s Yes Yea It 90 TcT To ColumnI Yea Yes Yea Yaa Yes Yes Yes -46 Group 1 Off Yea Yea Yea Yea Yaa Yea Yes Vaa Yes Yes Yea Yea Yea Yes Y'ea Yea Yea Yea Yaa Yea Yea Yes Yes Yea Yes Yea Yea Yes Yea 1= a7 Group Z On 1 Yea Yea Yes Yea Yea Yes Yea Yea 13 a0 TET To Coluan10 Yea Yea Yea Yea Yea Ysa Yea Is 29 BTcT To Cal 8 Yes Yes IS 90 Z < Yes Yes Yea Yea Tea Yea Yes 16 W8 TET To Column1 Yea Yea 17 a9 Group 2 Otf I Yea Yea Yea Yes Yea Yea - Yea 18 90 Group 3 On 10 Yea Yes t9 21 TET To Column8 Yea Yaa Yes Yea Yea Yea Yea 90 B~TET To Col a Yas Yea 21 -50 3 1 Yea Yea Yas Yea Yes Yes Yes t TET To ColuwnIS Yes Yes ~3 a5 Group 3 Oft 1 Yes Yea Yes Yea Yea Yea Yea 26 90 Matt 10 Yea Yes 19 6rouD 1 On 6 Yea Yes Yea Yea Yes Yea Yea 25 90 TET To Columna Yea Yea 27 -t6 B+TET To Col 1 Yea Yee Yea Yea Yes Yes Yea 2g sa7 1 I Yea Yea 29 90 TET To Column10 Yes Yea Yes Yea Yea Yea Yea 2A Group 1 Oft 8 Yea Yea 31 '9A Group 2 On L Yes Yea Yes Yaa Yes Yes Yea 32 -a8 TET To Coluwnt Yea Yes 33 a9 BaTET To Col I Yea Yea Yea Yea Yaa Yea Yea 3a 9A 2 10 Yes Yea Z1 TET To Column9 Yes Yea Yna Yea Yas Yaa Yea 36 9A Group 2 Off 4 Yea Yes 37 -58 Group 3 On t Yea Yes Yea Yaa Yea Yea Yes 38 TET To Column30 Yea Yes 39 +45 B~TET To Col 1 Yea Yea Yes Yes Yea Yes Yea a0 9A 3 IA Yea Yea at 19 TET To Coluwn8 Yea Yna Yea Yes Yea Yea Yea L2 9A ~6roup 3 Off a Yea Yea a3 -16 felt 1 Yes Yes Yaa Yea Yna Yea Yea Group t On Yea TET To Coluwn Yea Yes Yea Yea Yea Yaa B~TET To Cal Yes I Yea Yes Yea Yes Yes Yes TET To Coluwn Yea Group I Off Yes Yea Yea Yaa Yaa Yea Yea Yes Yaa Yea Yaa Yas Yea Yea Yea Yes Yea Yea Yea Yes Yea Yes Yaa Yes Yes Yas Yea Yes Yea Yea Yes Yea Yea Yea Yea Yes Yea Yea Yaa Yes Yea Yea Yea Yaa Ysa Ysa Yes Yea Yes Yea Yea Yea Yea YesYsa Yea Yes Yaa Yaa Yea Yea Yes Yas Yea Yea Yea Yea Yea Yes Yee Yes Yea Yea Yea Yea Yea Yna Yes Yes Yes Yaa Yea Yes Yas Yea Yes Yea Yas Yea Yes Yea tContinued next pape.) WO 93/13120 21 ~ ~ 9 2 8 PCT/US92/11165 STEP FUNCTION STEP STEP ACTIVE
SpF=
FOR
ARSES
NUMBER' 3 NHNE TiflE p _ _ -4i +d7 Sroup Z On - t Yea YeaYeaYeaYesYeaYes Y--_a 63 90 T=T To Calunn 10 Yea YesYeaYeaYeaYeaY-ssi_a Sd 20 B+TET To Col d Yea YeaYeaYesYtsYesYes Yca Z
i7 90 TET To Column d Yes YesYeaYeaYesYesYes Yea SA -dA 6reup 2 Off t Yea YeaYeaYeaYesYesYea Yea a9 +d9 6rouo 3 On I Yea YesYeaYeaYeaYeaYes Yea 60 90 TET To Column 10 Yes YeaYeaYeaYesYeaYea Yea 51 ~ B+TET To Col 8 Yea YesYeaYaaYesYeaYes Yes 21 3 ~
52 98 TET To Column d Yea YeaYeaYesYesYesYea Yea 53 -68 Group 3 Oft I Yaa YeaYasYeaYeaYeaYea Yes 5s a welt 30 Yea YeaYeaYeaYeaYeaYes Yea __ a5 Group I On I Yea YeaYeaYesYeaYeaYes Yes ~d 90 TET To Column 10 Vea YeaYeaYeaYeaYeaYea Yea ~7 13 6+TET To Col 3 Yea YesYeaYeaYesYeaYea Yes i ~d 90 TET To Column 6 Yea YeaYeaYaaYeaYeaYea Yes ~3 -a6 Group I Oft 1 Yea YeaYeaYeaYeaYeaYes Yes 60 +a7 Group .2 On 1 Yaa YesYeaYeaYeaYaaYes Yea 61 90 TET To Column I8 Yes YeaYeaYesYeaYeaYea Yes 62 20 B+TET To Col A Yea YesYasYaaYeaYeaYaa Yes 63 90 TET To Column a Yaa YeaYeaYaaYeaYeaYea Yes 61 -18 Group 2 Oft 1 Yes YeaYasYeaYaaYeaYea Yes 65 +a9 6rpup 3 On 1 Yaa YeaYesYasYesYeaYaa Yaa 66 90 T_T To Coluwn IB Yes YeaYasYeaYaa.YeaYes Vas 67 21 B+TET To Col B Yea YesYeaYaaYeaYesYea Yes 6A 98 TET To Coluwn a Yes YeaYeaYaaYeaYeaYea Yea 69 -SA Group 3 Otf I Yaa YeaYeaYesYasYaaYaa Yes 70 t felt 30 Yea YeaYeaYeaYeaYaaYea Yea 71 d6 Group I On 1 Yaa YaaYesYeaYeaYesYea Yas T_ 98 TcT To Column I8 Yea YeaYeaYeaYasYesYas Yes 73 19 B+TET Ta Col 8 Yea YeaYaaYesYesYasYaa Yes I
74 98 TET To Coluwn < Yaa YeaYeaYesYesYesYes Yas 75 -t6 Group 1 Oft 1 Yna YeaYeaYesYsaYeaYas Yes 76 '+a7 Group 2 On 1 Yea YeaYeaYsaYesYeaYea Yes 77 9A TET To Coluwn 10 Yas YeaYesYaaYaaYeaYes .Yea 78 2'D B~TET To Col B Yea YeaYeaYeaYaaYaaYea Yes 79 99 TET To Column t Yea YesYesYesYaaYeaYea Yes A8 -a8 Group 2 Off I Yas YaaYeaYaaYeaYesYaa Yea 81 ~ 6rouo 3 On t Yea YeaYaaYu YasYeaYea Yes +19 8Z 90 TET To Column ~ IA Yas YesYaaYeaYeaYea Yea Yaa -83 21 B~TET To Col B Yas YesYasYaaYea Yea 3. Yea Yea 8t 9A TET To Coluwn ' d Yea YeaYu YesYasYea Yes Yea BS -58 Group 3 Off I Ysa YeaYesYeaYeaYea Yea Yes 86 4 Hatt 30 Yea YesYeaYea Yes Yaa Yes Yea 87 15 Group t On 1 Yes YaaYesYaaYaa Yea Yaa Yea 88 90 TeT To Column 18 Yaa Yn>=
Yea Yea Yea Yea Yaa Yea (Continueo naa paqe.l WO 93/13120 212 4 9 2 8 PCT/US92/1116s iTEP FUHCTIOH STEP STEP RC1IVE , GRFc FOR
H M a NRHE T h R a ~ r 9 Ty~
99 !9 9+TET To Col - B Yea YesYeaYesYea Tes Yes 1 Yes 90 90 TET To Coluwn a Yea Y-_sYesYeaTes Yes Yes Yes 91 -ao Group I Off 1 Yes YesYeaYeaYea Yes Yes Yes 91 a7 Group Z On I Yea YesYesYeaYea Yes Yes Yea 93 9d TET To Coluwn 10 Yea YeaYeaYesYes Yea Yaa Yes 9a 20 6+TET To Col 8 Yea YeaYeaYeaYes Yea Yes Z Yea 95 90 TET To Coluwn a Yea YeaYesYeaYea Yea Yea Yea 95 ' Group 2 Otf 1 Yea YesYesYeaTea Vas Yes -a8 ~ Yea 97 +a9 6rouo 3 On 1 Yea YeaYesYeaYes Yea Yea Yea 99 9A TET To Coluwn 10 Yes YeaYeaYeaYes Yea Yea Yaa 99 21 8+TET To Col 8 Yea YeaYeaYeaYea Yes Yea 3 Yna 100 98 TET To Coluwn a Yea YeaYesYnaYaa Yea Yea Yes 101 -~d 6rou0 3 Off I Yea YeaYesYesYea Yaa Yes Yea 102 ~ Wait 30 Yas YeaYeaYeaYea Yes Yes Yea 103 +a5 Grpup t On 1 Yea YeaYesYeaYes Yes Yes Yea Ida 90 TET To Coluwn t0 Yes YeaYeaYeaYea Yea Yea Yes 105 19 6+TET To Col 6 Yea YeaYeaYeaYea Yna Yes I Yea 106 9A TET To Column 4 Yes YeaYeaYaaYna Yes Yes Yna 107 -a6 Group 1 Off I Yes YeaYeaYeaYna Yea Yes Yea 188 +a7 Group 2 On I Yaa YeaYeaYeaYea Yes Yaa Yaa 109 98 TeT To Coluwn 18 Yss YeaYeaYeaYea Yas Yes Yea 118 20 B+TET To Col 6 Yas YeaYesYeaYea Yes Yea 2 Yea 111 90 TET To Coluwn a' Yea YeaYesYeaYea Ysa Yas Yea I11 -aA Group 2 Off I Yaa YasYesYaaYea Yas Yes Yea 113 +a9 6rpup 3 On 1 Yea YesYeaYesYes Yea Yea Yea Ila 90 TET To Caluwn i0 Yaa YeaYeaYaaYna Yea Yes Yea 115 21 B+TET To Col B Yea YeaYesYeaYea Yea Yea 3 Yea 116 90 TET To Coluwn 4 Yes YesYeaYeaYaa Yea Yes Yaa 117 -~d Group 3 Off 1 Yes YesYeaYesYea Yea Yea Yes 118 a halt 3A Yne YaaYeaYasYes Yes Yea Yes I19 +a5 Group 1 On 1 Yea YeaYeaYaaYes Yaa Yea Yns 110 9A TET To Coluwn IA Ysa YeaYaaYesYns Yaa Yes Yea 121 ' B+TET To Col H Yas YeaYaaYeaYea Yes Yes 19 1 Yea 1ZZ 9i TET To Coluwn a Yea YnsYeaYeaYea Yaa Yas Yea 123 -a6 Group 1 Off I Yna YnaYnsYesYea Yas Yes Yea 126 +a7 Group Z On 1 Yes Yaa Yea Yes Yes Yns Yaa Yna IZS 90 TET To Coluwn 18 Yes Yna Yea.
Yaa Yes Yaa Yas Yaa 125 20 B+TET To Col H Yaa Yea 2 Yes Yea Yea Yna Yea Yea 127 9A TET To Coluwn ~ a Yna Yea Yes Yea Yea Yea Yaa Yea 12B -18 Group Z Off 1 Yea Yea Yaa Yea Yna Ysa Ysa Yes 129 +a9 Group 3 On t Yna Yea Ynn Yes Yea Yns Yes Yas 13A 9A TET To Coluwn IA Yna Yes Yaa Yaa Yea Yna Yas Yes 131 21 8+TET To Cal 8 Yea Yna 3 Yea Yea Yea Yes Yes Ysa 132 9A TET To Coluwn a Yas Yes Yes Yes Yes Yes Yas Yea 133 -5d 6reup 3 Off 1 Yas s a a Yes s YeL
Ye Ye Ye Ye Yes (Continuep neat peqe.l WO 93/13120 ~ ~ ~ ~ ~ ~ PCT/US92/11165 5TE? FUNCTION STEP STEP ACTIVE FOR 8R9E6 SAFE
.
N M s NRNE TIhE q r ' ~ 95i-??
~
134 1 Uett ~0 Yes Yea Yes Yep Yea Yes Yes Yes ~
Ij~ is Cao Preo 3 Yea Yes Yes Yea Yea Yea Yes Yes 136 10 318 To ~este 3 Yea Yes Yea Yes Yes Yes Y--_a Yea 137 ~ Reverse Fluan ~ Yes Yes Yes Yea Yes Yea Yes Yes 136 I Block Ftush d Yea Yes Yes Yea Yes Yea Yes Yea 139 91 Ceo To Coluwn 2: Yep Yes Yes Yea Yes Yea Yes Yes 140 10 i18To ~eate 3- Yea Yea Yes Yea Ysa Yea Yes Yea 141 4 IJeit 30 Yea Yes Yea Yea Yea Yea Yea Yes 142 ~ Reverse Flush S Yea Yes Yes Yea Yea Yea Y--_s Yaa 143 1 Block Flush 4 Yea Yea Yea Yes Yea Yea Yna Yes 144 61 SIS To ~eate 3 Yea Yea Yes Yea Yea Yea Yea Yes 145 13 s15 To Column ZZ Yea Yea Yes Yea Yea Yea Yea Yes 146 10 =I8 To ~aate ~ Yes Yea Yes Yea Yea Yea Yea Yea 157 4 deft 3A Yea Yea Yes Yes Yes Yea Yes Yea 1t6 2 Reverse Flusn ~ Yea Yea Yes Yea Yes Yea Yes Yes 149 I Block Flush 4 Yea Yea Yea Yea Yes Yea Yes Yes 150 9 s18 To Coluwn 10 Yea Yea Yes Yes Yaa Yea Yea Yea 151 34 FLuah to 4laateS Yes Yea Yes Yes Yns Yea Yea Yea 152 9 s18 To Coluwn 10 Yea Yes Yea Yes Yea Yea Yea Yaa 153 2 Reverse Flush S Yaa Yes Yea Yea Yea Yea Yes Yea 154 9 tIB To Coluwn 10 Yea Yea Yea Yes Yea Yea Yes Yes 155 Z Reverse Flush S Yes Yaa Yes Yaa Yea Yea Yea Yaa 9 S18 To Coluwn 10 Yea Yea Yea Yes Yes Yes Yes Yea 156 2 Reverse Flush 5 Yea Yea Yea Yes Yea Yea Yea 157 Yea 158 I Btoek Flush 4 Yes Yea Yea Yes Yea Yea Yea Yea 159 33 Cyela Elltry 1 Yas Yea Yaa Yes Yas Yes Yea Yes 160 6 ~este-?ort 1 Yea Yes Yea Yes Yes Yea Yes Yes 161 37 Relay 3 Pulse 1 Yea Yea Yea Yea Yea Yea Yes Yea 162 82 t14 To 4leate 3 Yes Yes Yea Yea Yna Yea Yes Yea 38 f17 To Yeate 3 Yea Yes Yes Yea Yes Yea Yea Yes 163 H S Yea Yea Yes Yes Yaa Yas Yea t Yea T
164 I0 ea e o i18 9 i18 To Coluwn 20 Yes Yes Yes Yes Yes Yea Yea Yea 165 ~ f17 To Coluwn 60 Yea Yea Yes Yea Yea Yea No 11 Yas 166 14 f11 To Coluwn 20 Yea Yes Yes Yes Yea Yes No 167 Yea 168 2 Ravaraa Flush 7 Yes Yea Yea Yea Yea Yea No Yea Coluwn l5 Yns Yea Yes Yes Yes Yea No t17 T Yea 169 11 o S Yea Yea Yes Yes Yea Yea No 170 34 Flush to W Yes ate 171 11 :17 To Coluwn IS Yea Yes Yaa Yaa Yea Yes wo ~ Yas 1n Z Revarae Flush S Yaa Yea Yaa Yaa Yas Yea Ho Yea 173 1a flt To Coluwn 28 Yaa Yes Yea Yes Yes Yea No Yea 174 34 Flush to Wate 10 Yes Yea Yes Yea Yea Yea No Yea 175 7 4eate-9ottle 1 Yes Yes Yaa Yas Yes Yes Yea Yea 176 9 a18 To Coluwn 10 Yes Yes Yes Yea Yes Yes Yea Yea sh 5 Yea Yes Yes Yea Yea Yea Yes Fl Yea 177 Z u 10 Yea Yea Yes Yea Yea Yea Yea Reverse Yea -178 9 s18 To Coluwn (Continued neat papn.>
5TE? FUNCTION STEP STEP RCTIVE FOR BRSES 9RFc N M Ft HRHE TIFIE A r [ r ~ ''7 STy~
179 '_ aeverae Flush _ ~ Yea Yes Yea Yes Yea Yes Yea Yes 180 ? s18 io Colunn 10 Yes Yes Yea Yes Yes Yes Yes Yes 191 2 Reverse Flusn S Yes Tea Yes Yes Yes Yes Yea Yes 182 I Bloek Fluan 3 Yes Yea Yea Yes Yea 'tea Yes Yes 2.24928 STEP' FUNCTION STEP iTP ACTIVE FOR BRfii S:,F_ HUhBER . T h R C C -x NRHE
I 10 318 To IJeate2 Yea Yes Yes Yea Yes Yea Yes Yea 3 =I8 Ta Column9 Yes Yea Yea Yea Yea Yes Yes 3 _ r7e~erae Fluani Yes Yea s I Block Fluah 3 Yea Yes Yea Yea Yea Yes Yes > > Apvanee FC 1 Yes Yes 6 - 2B Phca Prep 3 Yea Yes Yes Yes Yea Yea Yes 7 1S Group I On I Yea Yes 8 90 TT To Column 6 Yea Yea Yea Yea Yea Yea Yes 9 19 BTET To Col 6 Yes Yes 90 I 3 Yea Yea Yes Yea Yes Yea Yes II 1_ TET To Column~ Yea Yes I= 90 BTET To Col 3 Yes Yea Yea Yea Yes Yes Yes 13 19 I 3 Yea Yea I.t 3 TET To Column1 Yes Yea Yea Yaa Yea Yes Yea Ii -a6 9TET To Col 1 Yea Yes i6 47 t I Yea Yea Yea Yea Yea Yea Yea 17 10 s18 To Column6 Yes Y-es 18 I Group 1 Ott 3 Yes Yes Yes Yea Yea Yea Yea 19 98 Group 2 On 6 Yea Yea 28 20 s18 To ~aate 6 Yea Yes Yes Yea Yes Yes Yes 21 90 Block Flush 3 Yes Yes.
22 20 TET To Coluwn3 Yea Yes Yea Yea Yea Yes Yea 23 90 BtTET To Col 3 Yea Yes 2a 20 2 3 Yea Yes Yea Yea rea res Yea 25 9 TET To ColumnI Yea Yes 25 -a8 B~TET To Col 1 Yea Yes Yea Yes Yea Yea Yea 27 a9 Z 1 Yea Yea 28 10 Tai To ColumnL Yea Yea Yea Yea Yea Yea Yes 29 I 8flET To Col 3 Yea Yea 3B 90 2 6 Yea Yea Yea Yea Yea Yea Yes 31 ' s18 To Column6 Yaa Yea 32 21 Group 2 Otf 3 Yea Yea Yes Yes Yea Yea Yes 33 9A 'vroup 3 On 3 Yea Yea 3t 21 s18 To ~aate 3 Yes Yea Yes Yea Yea Yea Yea 35 9A Bloek Fluah 3 Vas Yea 36 2I TET To Coluwn1 Yes Yea Yea Yaa Yea Yea Yes 37 9 BaTET To Col ~ 1 Yes Yes 38 -Sa 3 20 Yes Yea Yea Yea Yes Ysa Yes 39 t TET To ColuwnS Yea 48 2 BtTET To Col 2 Yea Yaa Yea Yes Yaa Yea Yes al 10 3 9 Yea Yea 42 9 TET To ColumnS Yea Yes Yea Yea Yea Yaa Yea a3 2 9;TET To Col 3 Yaa Yes_ 10 3 Yea Yes Yea Yes Yes Yaa f18 To Coluwn Yea 6reup 3 Oft Yea Yes Yes Yea Yea Yes IJait Yea Reverse Fluah Yaa Yea Yea Yea Yea Yes s1A To Yaste Yes sib To Column Yaa Yea Yea Yaa Yea Yas Reverae Flusn Ysa s18 To ~aate Yea Yes.Yes Yea Yea Yea Yea Yea Yas Yes Yea Yea Yea Yea Yes Yaa Yaa Yes Yaa Yea Yea Yes Yes Yea Yea Yes Yea Yna Ysa Ysa Yes Yes Yea Yea Yea Yes Yea Yea Yea Yea Yes Yea Yea Yea Yes Yea Yea Yea Yes Yes Yea Yes Yaa Yea Yea Yea Yea Yea Yea Yaa Yea Yea Yea Yea Yea Yes Yu Yea Yas Yes Yes Yea Yea Yea Yas Yea Yea Yea Yea Yea Yea Yes Yes Yea Yea Yes Yes Yea Yee (Continued next pepe.l WO 93/13120 ~ ~ PCT/US92/11165 STEP FUNCTION STEP STEP FOR9RSE5 SRF=
RCTIV ~
NR11E T h p [ ~ SS Tj r ' t4 . 9loek Fluah - 3 Y -d~ 15 Group 1 On ~ Yes _ Y a a6 90 TET To Column o Yes Yes t7 19 9+TET To Col a Yes Yea 68 90 TET To Column 3 Yea Yes a9 t9 8+TET To Col 3 Yes Yea 50 9A TET To Column 3 Yes Yes 51 - 19 9+TET To Col 3 Yea Yea t ~
52 9 S18 To Column I Yea Yes 53 -t6 Group l Off I Yea Yea 54 +t7 Group 2 On I Yes Yes ' SIB To ~aate 6 Yea Yea 3 Yes Yes 56 1 6loek Fluah 57 90 TET To Column o Yea Yes 59 20 9+TET To Col 6 Yea Yes .3 90 TET To Colwn 3 Yes Yea 60 2A B+TET To Col 3 Yes Yes 61 90 TET To Coluwn 3 Yea Yes -e2 Z8 B+TET To Col 3 Yea Yes Yea Yea 63 9 S18 To Column 1 , Yea Ye 6s -t8 Group 2 Ott 1 a Yea Yea 65 +t9 Group 3 On 1 Yea Yes 66 18 S18 To haste t 3 Yes Yes 67 1 6loek Fluah Yes Yea 66 98 TET To Column 6 69 21 8+TET To Col 6 Yaa Yes Yes Yea 78 90 TET To Column 3 Yea 71 21 9+TET To Col 3 Yes 3 Yea Yes 72 98 TET To Colwn Yea 73 21 9+TE7 To Col 3 Yea Yea Yea 74 9 SIB 7o Column 1 Yea 75 '-SA Group 3 Ott I Yes Yes 28 Yes 76 l Wdt 3 Yea YeaYeaTeaYea Yes Yes Yea 77 16 Cap Prep 5 Yea YeaYeaYeaYea Yea Yes Yea 76 Z Reverse Flush Y YaaYea Yes Yea a Yes 79 1 HloeE Flush 3 Yaa Yeae I2 Yea YesVasYaaYes Yes Yas Yea 80 91 Cao To Colwn ~ Y Yea Yea 91 I0 i18 To haste 3 Yaa VasYesYeaea Yes 9 Yea YaaYeaYesYaa Yea Yes Yea 92 t halt ' S Yes YesYesYesYea Yae Yes Yea 93 Z Revarae Flush Y YeaYeaVas Yea Yes Yea 8t 81 i15 To liaate 3 Yea ea Yes 1A Yea YesYesYaaYea Yee Yes 95 13 S15 To Colwn a YeaYea Yes Yes Y Yea 96 t8 i18 To llsate 3 Yes Yeae 15 Yea YeaYeaYeaYea Yea Yes_ Yea A7 t ~eit ~ Yea YeaYeaYesYea Yea Yes Yes 88 Z Reverse Flush (Continueo next peqe.>
", WO 93/13120 2 1 2 4 9 2 $ pCl~L7S92/11165 STEP FUNCTION STEP STEP RCTIVE
SPF'c FOR
BRfiES
N h c s NPnE T M a 6 G 5 ST~o 0g ? SIB To Column - 9 Yna YesYeaYesYeaYesYns Yea ?0 3l Fiush to UeateS Yes YeaYesYesYesYeaYes Yes 91 ? 318 To Column 9 Yes YesYeaYesYesYeaYes Yea 92 2 Reverse Fluan 5 Yes YeaYesYeaYeaYeaYea Yes 93 3 118 To Column 9 Yea YeaYesYesYesYeaYea Yes 9a ~ Reverse Flusn 5 Yea YeaYeaYesYeaYeaYea Yea g5 I 3loek Flush 3 Yea YesYeaYesYnaYesYea Yes 96 33 Cyele Erttry I Yea YeaYeaYasYeaYeaYaa Yes 97 9 1t8 Ta Column S Yaa YeaYeaYeaYeaYesYea Yes 9g 2 Reverse Fluan S Yea YesYeaYeaYesYesYes Yea 5 ~eate-Port I Yea YesYeaYeaYeaYeaYea Yes _ 30 317 To ~eate 3 Yes YeaYeaYeaYeaYeaYea Yea 101 II t17 To Column 7 Yea YeaYeaYeaYesYeaYes No 102 3a FLuah to 4laate1 Yea YeaYesYesYeaYesYea No 103 11 t17 To Column 7 Yea YeaYea-YSaYesYesYea No 10a 3a FLuah to ~aateI Yea YesYeaYesYesYeaYes No 105 It t17 To C0lunn 7 Yaa YnaYeaYeaYeaYeaYns No 106 36 Flush to ~eateI Yaa YeaYeaYesYeaYeaYaa No 107 11 a17 To Column 7 Yas YesYeaYeaYeaYeaYea No 10B 3a Flush to hastet Yea YaaYeaYeaYaaYeaYas No 109 11 617 To Coluwn 7 Yas YeaYeaYaaYeaYesYea No 110 34 Flush to lute 1 Yea YnaYasYasYeaYaaYea No 111 11 t17 To Column 7 Yaa YeaYesYaaYaaYeaYna No 112 3a Flush to ~aate~S Yas YeaYesYaaYesYeaYaa No 113 9 318 To Column 9 Yea YeaYeaYaaYaaYaaYea No 116 3a Flush to Waste7 Yes YesYesYeaYesYeaYea No 115 7 Lleate-Bottle 1 Yea YeaYnaYeaYeaYeaYea Yes 116 9 t18 To Colwn 9 Yea YeaYesYeaYeaYesYea Yes t17 2 Reverse Flush S Yea YesYeaYeaYeaYesYes Yes IIB 9 118 To Column 9 Yea YeaYesYesYeaYeaYns Yes 119 2 Reverse Fluan S Yes YeaYesYeaYeaYeaYea Yea 120 1 Bloek Flush 3 Yea YeaYeaYeaYeaYesYea Yes W093/13120 ~ PCh/US92/II165 STEP FUNCTION STEP STEP RCTIVE FOR gR6E6 9RFE
N t1 3 NRHE ~ R ' ' STe?
I 10 i18 To ueste = Yes Yea Yes Yss Yes Yes Yes fes 2 9 SIB To Column9 Yes Yea Yea Yea Yes Yea Y._s Yes 3 Z Reverse Flusn~ Yea Yea Yes Yes Yes Yes Yes Yes a I glopk Flush 3 Yea Yes Yea Yea Yes Yea fes Yes 5 ACvenpe FC 1 Yea Yea Yea Yea Yes Yea Yes Yea 2B Phos Prep 3 Yes Yes Yea Yea Yea Yes Yea Yea 7 +a5 6rouo I On I Yea Yna Yes Yea Yes Yea Yes Yea 8 90 TET To Column6 Yea Yea Yaa Yaa Yaa Yea Yes Yea 9 19 8+TET To Cal 6 Yaa Yea Yes Yea Yea Yea.~YeaYes I
90 TET To Coluwn3 Yes Yea Yea Yea Yes Yea Yes Yaa II 19 8+TET To Col 3 Yes Yea Yes Yea Yea Yea Yea I Yea 13 90 T_T To Column3 Yea Yea Yea Yes Yea Yea Yes Yea 13 19 B+TET To Col 3 Yea Yea Yea Yes Ysa Yea Yes 1 Yes Ia 3 t18 To Coluwn1 Yea Yea Yea Yea Yea Yea Ye_s Yes -a6 firoup 1 Ott I Yea Yea Yaa Yea Yes Yes Yes Yea 16 +a7 Group 2 On 1 Yes Yea Yea Yes Yes Yea Yea Yaa 17 I0 SIB.To haste a Yea Yea Yea Yes Yea Yea Yes Yea IB 1 Bloek Flush 3 Yes Yes Yaa Yaa Yaa Yea Yaa Yes "" - 19 90 TET To Column6 Yea Yes Yea Yea Yaa Yea Yea Yea Z8 28 8+TE7 To Col 6 Yaa Yaa Yes Yaa Yea Yn Yea Z Yaa Z1 90 TET To Column3 Yes Yes Yea Yaa Yaa Yea Yes Yea 22 20 B+TET To Col 3 Yes Yea Yea Yea Ysa Yea Yes 2 Yes Z3 90 TET To Coluwn3 Yea Yes Yaa Yes Yes Yaa Yes Yes 2a 20 g+TET To Co1 3 Yes Yea Yea Yaa Yea Yea Yes 2 Yea 9 s18 To Coluwn1 Yes~Yea Yea Yaa Yea Yea Yea Yes Z6 -a8 Group 2 Ott 1 Yea Yea Yea Yes Yes Yea Yes Yea Z7 +a9 Group 3 On 1 Yea Yes Yea Yea Yea Yes Yes Yea 28 10 SIB To Wstn 4 Yaa Yes Yea Yea Yea Yea Yes Yea 29 I Bloek Flush 3 Yea Yea Yea Yea Yea Yes Yea Yes 98 TET To Coluwn6 Yea Yss Yes Yes Yes Yea Yea Yes 31 ' B+TET To Col 6 Yes Yaa Yea Yes Yea Yea Yes 21 3 Yea 32 9A 7ET To Coluwn3 Yea Yea Yea Yaa Yae Yea . Yes Yea 33 21 g+TET To Col 3 Yes Yaa Yas Yea Yea Yea Yes 3 Yea 3a 9A TET To Coluwn3 Yas Yes Yes Yaa Yaa Yaa Yes Yea ' 21 B+TET 1o Col 3 Yes Yea Yea Yea Yea Yea Yea 35. 3 Yes . 9 :18 To Coluwn1 Yea Yea Yea Yu Yes Yea Yea 36 Yas 37 -50 firoup 3 Ott t Yes Yea Yes Yea Yaa Yea Yes Yea Z0 Yes Yes Yea Yas Yas Yes Yes Yes 3g a Halt 3 Yea Yes Yea Yaa Yaa Yaa Yes Yaa 39 16 Cap Prep sh S Yea Yea Yaa Yes Yaa Yea Ycs Fl Yea Z u 3 Yea Yea Yea Yes Yes Yea Yes ai t Reverse Yes ek Flueh Bl a2 91 o 12 Yes Yes Yea Ysa Yaa Yes Yea To Coluwn Yes C
ap 3 Yes Yes Yes Yea Yea Yea Yes Yea a3 10 fib To sate (Canttnueo next pa0a.>
.,. W093/13120 PCT/US92/11165 STEP FUNCTION STEP STEP RCTIVE
9RF=
FOR
BRfiE6 ~
N M R 3 NAHE T h P ~ 3 ST~
ai 1 Uelt - 8 Yea YesYeaYesYeaYes Yes Yes a5 . Reverse Flusn5 Yes YesYeaYesYeaYes Yes Yea a6 91 SIS To uaate 3 Yea YeaYesYesYesYeaYea Yes a7 13 113 To Colunn10 Yes YeaYeaYeaYesYesYea Yes a8 i0 iI8 To ~este 3 Yea YesYesYeaYesYeaYea Yes 49 a fait I9 Yaa YesYeaYeaYeaYesYea Yea 90 2 Reverse Fiuah5 Yea YesYeaYesYeaYesYea Yes ~
51 9 i18 To Column9 Yea YeaYeaYeaYesYeaYea Yes 52 3a Flush to IJaateS Yea YesYeaYeaYeaYeaYea Yes 53 9 I18 To Column9 Yea YeaYeaYeaYaaYesYea Yes 5i Z Reverse Flush5 Yes YeaYesYesYesYea~YSa Yea 55 3 t18 To Column9 Yes YesYeaYeaYeaYeaYea Yea 50 2 Reverse FlushS Yea YeaYeaYesYesYeaYes Yes 57 1 BLock FLusn 3 Yes YeaYeaYeaYeaYeaYes Yea 58 33 Cyele Entry I Yea YeaYeaYaaYeaYesYen Yes 93 3 318 To Column9 Yea YesYesYeaYeaYeaYea Yea 60 '_ Revnrae Flush5 Yea YesYeaYeaYesYesYea Yea 61 6 ~este-Port t Yea YeaYeaYeaYeaYssYea Yea 62 30 i17 To ~eate 3 Yes YesYeaYeaYeaYeaYea Yea 63 11 ill To Column7 Yea YeaYeaYeaYesYeaYea No 6a 3a Flush to 4leste1 Yea YeaYeaYeaYesYesYea No 65 II i17 To Column7 Yes YesYeaYesYaaYeaYea No 66 3a Flush to 4esteI Yaa YeaYesYeaYesYesYea No 67 II t17 To Column7 Yes YeaYeaYeaYaaYeaYaa No 68 34 Flush to ~eate1 Yes YeaYeaYeaYesYaaYea No 69 II i17 To Column7 Yes YesYesYeaYeaYesYea No 70 3a FLuah to IJesteI Yea YesYeaYesYeaYesYes No 71 11 i17 To Column7 Yas YeaYeaYesYeaYeaYea No 71 3a Flush to IJeate1 Yea YeaYeaYesYesYeaYaa No 73 11 ti7 To Column7 Yea YesYeaYaaYeaYeaYes Na 7a 3a Flush to ~eateS Yes YeaYesYeaYeaYaaYea No i 9 i18 To Column9 Yea YesYeeYesYeaYeaYes No = ~ Flush to 4laate7 Yes YeaYeaYea Yea No 76 3a Yea Yea 77 7 fiesta-Bottle1 Yas YesYeaYes Yea Yea Yea Yea 7 9 ti8 To Column9 Yea Yes Yaa Yes Yea Yes Yea Yea 8 2 Reverse FlushS Yea Yes Yea Yes 79 Yes Yea~Yea Yea 9 t18 To Column9 Yea Yea Yes Yea Yea Yea Yea Yea 80 2 Reverse FLuahS Yas Yes Yaa Yea HI Yes Yea Yea Yes 82 I Bloek Flush 3 Yea Yea Yea Yns Yea Yes Yea Yes 5$
NUMBER a NH11E -TIRE 9 ~ ~ T 5 5 -' T«
I 10 t18 To 4lsate 2 Yea YeaYeaYeaYeavesYes Yea 9 SIA To Column IS Yes YesYesYeaYeaYesYes Yes 3 ? Reverse Flush 20 Yea YesYeaYeaYesYesYes Yea a I Bloek FLush 6 Yes YeaYeaYeaYeaYesYea Yes I6 Cao Preo 10 Yea YeaYeaYeaYesYeaYea Yes a - . 30 Yea YeaYeaYeaYaaYeaYes Yes 91 Ceo To Column 7 10 a18 To ~aate 3 Yea YesYeaYeaYeaYesYea Yea B t Block Flush ~ Yes YeaYeaYaaYeaYeaYea Yes 9 4 Bait 300 Yea YesYeaYeaYeaYes.YeaYes I6 Cao Preo i0 Yea YeaYesYeaYeaYeaYea Yea 11 91 Cao To Column 30 Yes YesYeaYeaYeaYeaYea Yes I~ 10 i16 To Ueste~ 3 Yes YeaYeaYeaYeaYesYes Yes 13 I Bloek Fluan a Yea YesYeaYeaYeaYeaYea Yea la a Ueit 300 Yea YeaYesYeaYesYeaYes Yes 2 Reverse Fluan 10 Yea YeaYesYeaYeaYeaYea Yes 16 10 tIB To haste. 3 Yea YaaYnaYnaYeaYeaYea Yes 17 9 :18 To Column 15 Yea YaaYeaYeaYesYeaYes Yaa 18 2 Reverse Flush IA Yes YeaYesYesYaaYesYea Yes i9 9 t18 To Column IS Yna YeaYesYeaYeaYeaYea Yes 2 Reverse Flush t0 Yes YeaYesYeaYeaYnaYea Yea 21 9 it8 To Column 15 Yes YaaYnsYaaYeaYeaYes Yes 22 2 Reverse Flush I0 Yes YeaYeaYsaYeaYeaYaa Yea 23 9 t18 To Column 15 Yea YesYesYeaYesYeaYes Yas 26 2 Reverse Flush IB Yea YeaYeaYesYesYeaYea Yea 9 t18 To Column 15 Yea YeaYeaYeaYeaYeaYea Yes 25 2 Reverse Flush 6B Yes YeaYeaYeaYeaYesYes Yes Z7 I Bloek Fluan 5 Yea YesYeaYeaYesYeaYea Yea .... W093/13120 PCT/US92/11165 212492$
STEP FUNCTION STEP STEP RCTIVE SRFc FOR
BRSES
l9ER E NR11E TZhE R 6 C T c c ~ STc?
N
, S(j I 2 Reverse Flush60 Yes YeaYesYeaYeaYeaYea Yes Z Z7 i10 To CollectI7 Yea YeaYeaYaaYeaYeaYea Yes 3 10 SIB To 4laateS Yes YesYeaYeaYesYesYea Yes I Block Flush S Yea YeaYeaYaaYesYeaYea Yes S 6 4lelt 660 Yas YeaYeaYeaYeaYeaYea Yea 0 27 ti0 To Collect, Yea YesYeaYeaYeaYeaYes Yea ~~18 7 I0 SIB To ~aate S Yea YeaYesYesYeaYesYea Yea 8 1 Block Fluan S Yes YasYeaYaaYeaYeaYea Yea 9 ~ ~at,t 660 Yea YesYesYaaYesYeaYaa Yea 27 il0 To Collect18 Yes YesYeaYnaYeaYeaYes Yea II IB SIB To Wate S Yes YeaYeaYeaYeaYea'Yea Yea 1. I Block Fluan ~ Yea YeaYeaYesYesYesYes Yea IS t gait 660 Yea YesYeaYesYeaYesYea Yea la 27 i10 To Collect17 Yes YeaYesYeaYesYesYea Yea IS 10 i18 To ~aate S Yea YeaYeaYeaYeaYesYes Yea I6 t Block Flush S Yea YasYeaVaaYesYesYea Yea 17 s uut 660 Yes YeaYesYeaYaaYeaYea Yes IB 0 Flush To CLOT9 Yea YaaYeaYeaYeaYeaYaa Yes 19 Z7 i10 To Collectt4 Yea YeaYeaYeaYeaYeaYea Yea B Fluan To CLOT9 Yea YaaYeaYeaYeaYesYea Yea ZI . Raverae Flush60 Yes YeaYeaYesVaaYeaYes Yes 2Z 1 Block Flush Yaa YeaYaaYeaYeaYeaYea Yea Z5 10 i18 To Waste S Yaa YeaYaaYeaYaaYeaYea Yea Zi 9 SIB To Column30 Yea YeaYeaYeaYaaYeaYea Yes ZS 2 Reverse Flush60 Yna YasYeaYnaYaaYesYea Yea ZS I Fluan 18 Yea YesYesYeaYeaYesYes Yea 8loek . 2 Yea YeaYeaYeaYeaYeaYea Yea Z7 6Z s10 Vent WO 93/13120 PCT/US92/1 t 165 ~~~~~2g STEP FUNCTION STEP STEP RCTIV SRFE
FOR
i NR11E ThE R [ r 6 7 STEP
5-.
I '_8 Phoa Prec 10 Yes YeaYesYesYeaYesYea Yes 52 A To IJeate S Yes YesYeaYes~YesYeaYea Yes 3 .3 o To IJeate S Yea YeaYeaYeaYeaYeaYea Yes 6 56 C To 4leate 5 Yea YeaYeaYesYeaYeaYea Yes 5 E5 T To 4leste S Yea YeaYeaYeaYesYesYes Yea 6 ~ SS To Ylaate S Yea YeaYeaYeaYesYesYea Yea 7 57 36 To W ste S- Yea YeaYeaYesYesYeaYes Yes 8 58 Z7 To ~aate 6 Yea YesYesYeaYeaYeaYea Yes 9 61 TET To ~aate B Yea YeaYeaYesYeaYesYes Yes 10 10 i18 To ~este I0 Yes YesYeaYeaYesYeaYea Yes 11 Ifi Ceo Prep 10 Yes YesYesYeaYeaYeaYea Yes 1'_ .3 Cao A To ~aate5 Yea YeaYeaYesYesYeaYea Yea I~ 60 Cao 8 To hasteS Yes YeaYesYeaYes'feaYea Yes ii A1 SIS To ~eate 8 Yea YeaYeaYeaYesYeaYes Yes 15 A2 it1 To ~este 8 Yes YeaYeaYeaYeaYesYea Yes I6 30 i17 To ~aate 10 Yes YeaYeaYea.YeaYesYea- Yes 17 18 i18 To Weate 15 Yea YsaYeaYeaYesYesYea Yea 18 1 8loek Fluah IS Yea YeaYeaYeaYesYesYea Yes WO 93/13120 ~ ~ 2 4 9 2 8 PCT/US92/11165 6'- 6bT 6TT Tb6 TT6 TT6 TF6 TT6 TT6 TT6 TT6 TT6 TT6 TT6 TT6 TT6 TT6 TT6 TTT TTT TTT TTT TTT .TTT TT -3' VERSION Z.00 SEQUENCE NRIIE:t S%-2 SEQUENCE LEH6TM:10 OATE: Au0 27.
TI!: ta:06 COHHENT:
S'- ?7T bAC T65 T -3
The portion of the comb body through the 15 (TTX') repeats is first synthesized using 33.8 mg ,. WO 93/13120 PCT/US92/11165 a- aopropyl-derivatized thymidine controlled pore glass (CPG) (2000 A, 7.4 micromoles thymidine per gram support) with a 1.2 cycle protocol. The branching site nucleotide was of the foxacula:
O
where R2 represents O
For synthesis of the comb body (not including sidechains), the concentration of beta cyanoethylphosphoramidite monomers was 0.1 M for A, C, G
WO 93/13120 12 r~ 9 2 g PCT/US92/11165 and T, 0.15 M for the branching site monomer E, and 0.2 M
for Phostel° reagent. Detritylation was done with 3t trichloroacetic acid in methylene chloride using stepped flowthrough for the duration of the deprotection. At'the conclusion the 5' DMT was replaced with an acetyl group.
Cleavable linker R and six base aidechain extensions of the formula 3'-RGTCAGTp (SEQ ID N0:1) were synthesized at each branching monomer site as follows.
The base protecting group removal (R2 in the formula above) was performed manually while retaining the CPG
support in the same column used for synthesizing the comb body. In the case of R2 - levulinyl, a solution of 0.5 M
hydrazine hydrate in pyridine/glacial acetic acid (1:1 v/v) was introduced and kept in contact with the CPG
support for 90 min with renewal of the liquid every 15 min, followed by extensive washing with pyridine/glacial acetic acid (1:1 v/v) and then by acetonitrile. After the deprotection the cleavable linker R and nix baae sidechain extensions were added using a 6.4 cycle.
In these syntheses the concentration of phosphoramidites was 0.1 M (except 0.2 MR and Phostel°
reagent; R was 2-(4-(4-(2-Dimethoxytrityloxy)ethyl-)phenoxy 2,3-di(benzoyloxy)-butyloxy)phenyl)ethyl-2-cyanoethyl-N,N-diisopropylphosphoramiditel.
Detritylation is effected with a solution of 31< trichloroacetic acid in methylene chloride using continuous flowthrough, followed by a rinse solution of toluene/chloromethane (1:1 v/v). Branched polynucleotide chains were removed from the solid supports automatically in the 380H using the cycle ~CE
NH3.~ The ammonium hydroxide solution was collected in 4 ml screw-capped Wheaton vials and heated at 60°C for 12 hr to remove all base-protecting groups. After cooling to room temperature the solvent was removed in a Speed-Vac''N' evaporator and the residue dissolved in 100 ~1 water.
3' backbone extensions (segment A), sidechaia extensions and ligation template/linkers of the following structures were also made using the automatic ' synthesizer:
3' Backbone extension 3'-TCCGTATCC'fGGGCACAGAGGTGCp-5' (SEQ ID N0:2) Sidechain extension 3'-GATGCG(TTCATGCTGTTGGTGTAG)3-5' (SEQ ID N0:3) Ligation template for linking 3' backbone extension 3'-AAAAAAAAAAGCACCTp-5' (SEQ ID N0:4) Ligation tem-plate for link-ing sidechain extension 3'-CGCATCACTGAC-5' (SEQ ID NO: S) r The crude comb body was purified by a standard polyacrylamide gel (7% with 7 M urea and 1X TBE zunning buffer) method.
The 3' backbone extension and the sidechain~
extensions were ligated to the comb body as follows. The comb body (4 pmole/~1), 3' backbone extension (6.25 pmole/~1), sidechain extension (93.75 pmole/~1), sidechain linking template (75 pmoles/~1) and backbone linking template (5 pmole/P1) were combined in 1 mM ATP/
5 mM DTT/ 50 mM Tris-HC1, pH 8.0/ 10 mM MgCl2/ 2 mM
spermidine, with 0.5 units/W1 T4 polynucleotide kinase.
The mixture was incubated at 37°C for 2 hr, then heated in a water bath to 95°C, and then slowly cooled to below 35°C over a 1 hr period. 2 mM ATP, 10 mM DTT, 14%
polyethylene glycol, and 0.21 units/P1 T4 ligase were added, and the mixture incubated for 16-24 hr at 23°C.
The DNA was precipitated in NaCl/ethanol, resuspended in water, and subjected to a second ligation as follows.
..,~... .. The mixture was adjusted to 1 mM ATP, 5 mM DTT, 14%
polyethylene glycol, 50 mM Tris-HC1, pH 7.5, 10 mM MgCl2, 2 mM spermidine, 0.5 unita/~1 T4 polynucleotide kinase, and 0.21 units/~1 T4 ligase were added, and the mixture incubated at 23°C for 16-24 hr. Ligation products were then purified by polyacrylamide gel electrophoresis.
After ligation and purification, a portion of the product was labeled with 32P and subjected to cleavage at the site of R achieved by oxidation with aqueous NaI04 for 1 hr. The sample was then analyzed by PAGE to determine the number of sidechain extensions incorporated by quantitating the radioactive label in the bands on the gel. The product was found to have a total of 45 labeled probe binding sites.
.-.. WO 93/13120 212 ~ 9 2 ~ PCT/US92/t t 165 Hybridization Assav for HHV DNA
A "15 X 3" amplified solution phase nucleic acid sandwich hybridization assay format was employed in this example. The "15 x 3" designation derives from the fact that the format employs two multimers: (1) an amplifier probe having a first segment (A) that binds to HHV nucleic acid and a second segment (H) that hybridizes to (2) an amplifier multimer having a first segment (H~) that hybridizes to the segment (H) and fifteen iterations of a segment (C), wherein segment C hybridizes to three labeled oligonucleotides.
The amplifier and capture probe segments and their respective names used in this assay were as follows.
H6V Amplifier Probes HHV.104* (SEQ ID N0:6) HHV.94~ (SEQ ID N0:7) CCTRCTCGTGTTACAGGCGGGGTTTTTCTT
HBV.76* (SEQ ID N0:8) TCCATGGCTGCTAGGSTGTRCTGCCAACTG
HBV.87~ (SEQ ID N0:9) GCYTAYAGACCACCAAATGCCCCTATCYTA
HBV.45~ (SEQ ID NO:10) CTGTTCAAGCCTCCAAGCTGTGCCT'1GGGT
HHV.93~ (SEQ ID NO:11) CATGGAGARCAYMACATCAGGATTCCTAGG
HHV.99~ (S~Q ID N0:12) TCCTGGYTATCGCTGGATGTGTCTGCGGCGT
FIHV.78~ (SEQ ID N0:13) GGCGCTGAATCCYGCGGACGACCCHTCTCG
HSV.81~ (SEQ ID N0:14) C'ITCGCTTCACCTCTGCACGTHGCA1C~
HHV.73~070590-C (SEQ ID N0:15) GGTCTSTGCCAAGTGTTTGCTGACGCAACC
HBV.77~070590-b (SEQ ID N0:16) CCTRCGCGGGACGTCCTTTGTYTACGTCCC
HHV.D44~070590-A (SEQ ID N0:17) , '' MCCTCTGCCTAATCATCTCWT'GTWCATGTC
HHV.79~ (SEQ ID N0:1B) CGACCACGGGGCGCACCTCTCTTTACGCGG
HHV.82~ (SEQ ID N0:19) TGCCCAAGGTCTTACAYAAGAGGACTCTTG
HHV.7lt (SEQ ID N0:20) CGTCAATCTYCRCGAGGACZGGGGACCCTG
HBV.102~ (SEQ ID N0:21) ATGTTGCCCGTTTGTCCTCTAMTTCCAGGA
HHV.lOlt (SEQ ID N0:22) ATC'iTCTTRTTGGTTCTTCTGGAYTAYCAA
HBV.100~ (SEQ IDN0:23) ATCATMTTCCTCTTCATCCTGCTGCTATGC
HHV.98~ (SEQ ID N0:24) CAATCACTCACCAACCfCYTGTCCTCCAAY
HHV.97t (SEQ ID N0:25) GTGTCYTGGCCAAAATTCGCAGTCCCCAAC
HHV.96~ (SEQ ID N0:26) CTCGTGGTGGACTTCTCTCAATTTTCTAGG
HHV.95~ (SEQ ID N0:27) GACAAGAATCCTCACAATACCRCAGAGTCT
HHV.92~ (SEQ ID N0:28) TTTTGGGGTGGAGCCCRCAGGCTCAGGGCR
HBV.91~ (SEQ ID N0:29) CACCATATTCIZGGGAACppGARCTACAGC
HHV.88~ (SEQ ID N0:30) A~CTTCCGG~ACTAC'IGT'IGTTAGACGA
HHV.86t (SEQ ID N0:31) GTVTCITTYGGAGTGTGGATTCGCACTCCT
HHV.D47t (SEQ ID N0:32) ,... W093/13120 212 4 ~ 2 f3 PCT/US92/11165 HBV.D46. (SEQ ID N0:33) TTTGGGGCATGGACATYGAYCCRTATAAAG
HHV.BS (SEQ ID N0:34) AAWGRTCTTTGTAYTAGGAGGCTGTAGGCA
HHV.84 (SEQ ID N0:35) RGACTGGGAGGAGYTGGGGGAGGAGATTAG
HBV.83 (SEQ ID N0:36) CCTTGAGGCMTACTTCAAAGACTGTRTGTT
HHV.80 (SEQ ID N0:37) GTCTGTGCCTTCTCATCTGCCGGWCCGTGT
HHV.75* (SEQ ID N0:38) AGCMGCTTGTTTTGCTCGCAGSMGGTCTGG
HHV.74 (SEQ ID N0:39) GGCTCSTCTGCCGATCCATACTGCGGAACT
HBV.72* (SEQ ID N0:40) MTRAACCTiTACCCCGTTGCTCGGCAACGG
HHV.51 (SEQ ID N0:41) GTGGCTCCAGTTChGGAACAGTAAACCC2G
HHV.67 (SEQ ID N0:42) RAARCAGGCTTTYACTTTCTCGCCAACTTA
HBV.70* 062890-A (~3Q ID N0:43) CCTCCRCCTGCCTCYACCAATCGSCAGTCA
HHV.65 (SEQ ID N0:44) HHV Capture Probes HBV. 60'(SEQ ID N0:45) TATTCCCATCCCATCYTCCIGGGCZ'ITCGS
HHV. 64 (SEQ ID N0:46) TATATGGATGATGTGGTATTGGGGGCCAAG
HHV. 63'(SEQ ID N0:47) CGTAGGGCTTTCCCCCACTGTTTGGCTTTC
HHV. 62 (SEQ ID N0:48) GCTCAGTTTACTAGTGCCATTTGTTCAGTG
HHV. 61 (SEQ ID N0:49) _ CCTATGGGAGRGGGCCTCAGYCCGTTTCTC
HHV.89~ (SEQ ID NO: SO) ' GTCCCCTAGAAGAAGAACTCCCTCGCCTCG
HBV.90~ (SEQ ID NO:51) ACGMAGRTCTCMATCGCCGCGTCGCAGAAGA
HBV.D13~ (SEQ ID N0:52) . ~TCT~~TCTCJiATGTTAGTATYCC
HBV.D14~ (SEQ ID N0:53) GACTCATAAGGTSGGRAACTTTACRGGGCT
ooh ~Plifier probe contained, in addition to the sequences substantially complementary to the HBV
sequences, the following S' extension complementary to a segment of the amplifier multimer, AGGCATAGGACCCGTGTCTT (SEQ ID N0:54).
Each capture probe contained, in addition to the sequences substantially complementary to HHV DNA, the following downstream sequence complementary to DNA bound to. the solid phase (i.e, complementary to XT1~), CTTCITTGGAGAAAGTGGTG (SEQ ID NO:SS).
Microtiter plates were prepared as follows.
White Microlite 1 RemovawellT"~ strips (polystyrene microtiter plates, 96 wells/plate) were purchased from Dynatech Inc. Each well vas filled with 200 ~1 1 N HC1 and incubated at room temperature for 15-20 min. The plates were then washed 4 times with 1X PBS and the wells aspirated to remove liquid. The wells were then filled with 200 P1 1 N NaOH and incubated at room temperature for 15-20 min. The plates were again washed 4 times with 1X PHS and the wells aspirated to remove liquid.
Poly(phe-lys) was purchased from Sigma Chemicals, Inc. This polypeptide hoe a 1:1 molar ratio of phe:lys and an average m.w. of 47,900 gm/mole. It has an average length of 309 amino acids and contains 155 amines/mole. A 1 mq/ml solution of the polypeptide was mixed with 2M NaCl/1X PHS to a final concentration of 0.1 mg/m1 (pH 6.0). 100 p1 of this solution was added to each well. The plate was wrapped in plastic to prevent dzying and incubated at 30°C overnight. The plate was then washed 4 times with 1X PHS and the wells aspirated to remove liquid.
The following procedure was used to couple the oligonucleotide XT1~ to the plates. Synthesis of XT1~
was described in EPA 883096976. 20 mg diauccinimidyl suberate was dissolved in 300 ~1 dimethyl fozaamide (DMF). 26 OD260 unite of XT1~ was added to 100 ~1 coupling buffer (50 mM sodium phosphate, pH 7.8). The coupling mixture was then added to the DSS-DMF solution and stirred with a magnetic stirrer for 30 mia. An NAP-25 column was equilibrated with 10 sM sodium phosphate, pH 6.5. The coupling mixture DSS-DMF solution was added to 2 ml 10 mM sodium phosphate, pH 6.5, at 4°C.
The mixture was vortexed to mix and loaded onto the equilibrated NAP-25 column. DSS-activated XTl~ DNA was eluted from the column with 3.5 m1 10 mM sodium phosphate, pH 6.5. 5.6 OD260 units of eluted DSS-activated XT1~ DNA was added to 1500 m1 50 mM sodium phosphate, pH 7.8. 50 ~1 of this solution was added to each well and the plates were incubated overnight. The plate was then washed 4 times with 1X PHS and the wells aspirated to remove liquid.
Final stripping of plates was accomplished as follows. 200 ~L of 0.2N NaOH containing O.St (w/v) SDS
was added to each well. The plate was wrapped in plastic and incubated at 65°C far 60 min. The plate was then washed 4 times with 1X PHS and the wells aspirated to remove liquid. The stripped plate was stored with desiccant beads at 2-8°C.
g WO 93/13120 12 ~ g 2 g PCT/US92/11165 Sample preparation consisted of delivering 12.5 ~1 P-R buffer (2 mg/ml proteinase R in 10 mM Tris-HC1, pH 8.0/ 0.15 M NaCl/ 10 mM EDTA, pH 8.0/ 1t SDS/ 40 ~g/ml sonicated salmon sperm DNA) to each well.
A standard curve of HsV DNA was prepared by diluting cloned HHV, subtype adw, DNA in HHV negative human serum and delivering aliquots of dilutions corresponding co 1000, 3000, 10,000, 30,000, or 100,000 molecules to each well. Tests for cross-hybridization to heterologoua DNAs were done by adding either purified DNA or infected cells to each well. Amounts for each organism are indicated in the Table.
Plates were covered and agitated to mix samples, then incubated at 65° C to release nucleic acids.
A cocktail of the HHV-specific amplifier and capture probes listed above was added to each well (5 fmoles of each probe/well, diluted in 1 N NaOH). Plates were covered and gently agitated to mix reagents and then incubated at 65° C for 30 min.
Neutralization buffer was then added to each well (0.'7'7 M 3-(N-moipholino)propane aulfonic acid/1.895 M NaCl/0.185 sodium citrate). Plates were covered and incubated for 12-18 hr at 65° C.
After an additional 10 min at room temperature, the contents of each well were aspirated to remove all fluid, and the wells washed 2X with washing buffer (0.1t SDS/0.015 M NaCl/ 0.0015 sodium citrate).
Amplifier muitimer was then added to each well (30 fmoles/well). After covering plates and agitating to mix the contents in the wells, the plates were incubated for 30 min at 55° C.
After a further 5-10 min period at room temperature, the wells were washed as described above.
WO 93/13120 2 1 ? 4 ~ 2 $ PCT/US92/11165 Alkaline phosphatase label probe, disclosed in EP 883096976, was then added to each well (40 ~1/well of 2.5 fmoles/~1). After incubation at 55°C for 15 min, and min at room temperature, the wells were washed twice as S above and then 3X with 0.015 M NaCl/0.0015 M sodium citrate.
An enzyme-triggered dioxetane (SChaap et al., Tet. Lett. (1987) 28:1159-1162 and EPA Pub. No. 0254051), obtained from Lumiqen, Inc., was employed. 20 ~1 Lumiphos 530 (Lumigen) was added to each well. The wells were tapped lightly so that the reagent would fall to the bottom and gently swirled to distribute the reagent evenly over the bottom. The wells were covered and incubated at 37°C for 40 min.
Plates were then read on a Dynatech ML 1000 luminometer. Output was given as the full integral of the light produced during the reaction.
Results from an exclusivity study of the HHV
probes is shown in the Table below. Results for each standard sample are expressed as the difference between the mean of the negative control plus two standard deviations and the mean of the sample minus two standard deviations (delta). If delta is greater than zero, the sample is considered positive. These results indicate the ability of these probe sets to distinguish HHV DNA
from heterologous organisms and a sensitivity of about 1000-3000 H8V molecules.
Table Sample Amount Delta HHV 1 X 105 25.99 HHV 3 X 104 6.51 HBV 1 X 104 3.00 HHV 3 X 103 0.93 HBV 1 X 103 -0.20 Control __ H~ ~ 8 X i05 -0.39 ~1 3 .3X 106 -0.48 HTLV-II2 1 X 105 -0.07 HT'LV-I2 1 X 105 -0.23 HIV 1 X 107 -0.31 pBR325 1 X 107 -0.27 Streptococcus sanguis 1 X 107 -0.31 Streptococcus pyogenes 1 X 107 -0.36 Streptococcus pneumoniae 1 X 107 -0.38 Streptococcus fecalis 1 X 107 -0.28 Streptococcus agalactiae 1 X 107 -0.26 Streptococcus epidermidis 1 X 107 -0.31 Staphylococcus aureus 1 X 107 -0.34 Serratia marceacena 1 X 107 -0.30 Pseudomonas aeruginosa 1 X 107 -0.23 Proteua mir3bilis 1 X 107 -0.43 Peptostreptococcus 1 X 107 -0,46 anerobius Lactobacillus acidophilus 1 X 107 -0.33 Rlebaiella pneumoniae 1 X 107 -0.12 Haemophilus influenza 1 X 107 -0.34 Escherichia coli 1 X 107 -0.44 Enterobacter aerogenea 1 X 107 -0.23 Mycobacterium leprae 1 X 107 -0.18 1 denotes pfu in infected cells 2 denotes proviral copies Modifications of the above-described modes for carrying out the invention that are obvious to those of skill in biochemiatzy, nucleic acid hybridization assays, and related fields are intended to be within the scope of the following claims.
WO 93/13120 ~ PCT/US92/11165 _27_ SEQUENCE. LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Irvine, Bruce D.
Rolberg, Janice A.
Running, Joyce A.
Urdea, Michael S.
(ii) TITLE OF INVENTION: HBV PROBES FOR USE IN SOLUTION
PHASE SANDWICH HYBRIDIZATION ASSAYS
(iii) NUMBER OF SEQUENCES: 55 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Morrison ~ Foerster (H) STREET: 755 Page Mill Road (C) CITY: Palo Alto (D) STATE: California (E) COUyTRY: USA
(F) ZIP: 94304-1018 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (H) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: 07/813,586 (H) FILING DATE: 23-DEC-1991 (C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Thomas E. Ciotti (H) REGISTRATION NUMBER: 21,013 (C) REPERfiNCE/DOCRET NUMBER: 22300-20234.00 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 415-813-5600 (H) TELEFAX: 415-494-0792 (C) TELEX: 706141 (2) INFORMATZON FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ZD NO:1:
TGACIGFL
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs (B) TYPE: nucleic acid (C) STRANDEDNBSS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
GATGTGGTTG TCGTACTTGA TGTGG!'1GTC GTACIZGATG TGGTTGTCGT AC11GCGTAG 60 (2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs (H) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
(2) INFORMATION FOR SEQ ID N0:5:
(i> SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 base pairs WO 93/13120 ~ PCf/US92/11165 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear 5 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: S:
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs 10 (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
TTGTG('aGTCT 2TIGGGYTIT GC'fGCYCCWT 30 (2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid 20 (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
CCrICCTCGT'G TTACJVGGCGG GGTSTITCI1' 30 (2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUBNCB CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single 30 (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
TCCAT'GGCTG CTAGGSI~,lR CTGCCAAClG 30 35 (2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
(2) INFORMATION FOR SEQ ID N0:10:
10 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:10:
CTGTTCJ1AGC CTCCAAGCfG 1GCCT~GGGT' 30 (2) INFORMATION FOR SEQ ID N0:11:
(i) SEQUENCE CHARACTERISTICS:
20 (A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear 25 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
(2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCB CHARACTERISTICS:
(A) LENGTH: 31 base pairs 30 (H) 'TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
WO 93/13120 1 2 ~ 9 2 g PCT/US92/11165 (2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:
GGCGC'TGAAT CCYGCGGACG ACCCBTCTCG 30 (2) INFORMATION FOR SEQ ID N0:14:
(i1 SEQUENCfi CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:
CT1'CGCTTCA CCTC1~GCACG TFIGCA1'GQ~Ti 30 (2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCB CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:15: ' GG1'CfST'GCC AAGTG'ITIGC TGACGCAACC 30 (2) INFORMATION FOR SEQ ID N0:16:
(i1 SEQUENCE CHARACTERISTICS:
(A) LfiNGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDHDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCfi DESCRIPTION: SEQ ID N0:16:
CC17CCGCGGG ACGTCCITiG TYTACGTCCC 30 (2) INFORMATION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:
MCCTCTGCCT AATCATCTC4I TGTSiCATGTC 30 (2) INFORMATION FOR SEQ ID N0:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:18:
(2) INFORMATION FOR SEQ ID N0:19:
(i) SEQUENCB CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ZD N0:19:
~'CCCAAGGT C1TACAYAAG 71GGACfC'ITG 30 (2) INFORMATION FOR SEQ ID N0:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs -(H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear WO 93/13120 1 ~ ~ 9 2 g PCT/US92/11165 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:20:
CGTCAATCTY CKCGAGGALT GGGGACCCfG
~ 37 5 (2) INFORMATION FOR SEQ ID N0:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:21:
ATGTTGCCCG TTTGTCC'f'CT AM! I'CC~,C,p (2) INFORMATION FOR SEQ ID N0:22:
15 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (Dl TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:
ATCTTCTTRT TGGTTCTTCT GGAYTAYCAA
(2) INFORMATION FOR SEQ ID N0:23:
(i) SEQUENCE CHARACTERISTICS:
25 (A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear 3p (xi) SEQUENCE DESCRIPTION: SEQ ID N0:23:
(2) INFORMATION FOR SEQ ID N0:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid WO 93/13120 2 1 2 4 9 2 8 P~/US92/11165 (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:24:
CAATCACTCA CCAACCfCYT GTCCTCCAAY 30 (2) INFORMATION FOR SEQ ID N0:25:
(i1 SEQUENCfi CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid to (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:25:
GTGTCYT'GGC CAAAATTCGC AGT'CCCCWC 30 (2) INFORMATION FOR SEQ ID N0:26:
(i) SEQUENCB CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNBSS: single (D) TOPOLOGY: linear (xi) SEQUENCB DfiSCRIPTION: S8Q ID N0:26:
CTCGTGGTGG ACTCCTCrCA ATTTTCTAGG 30 (2) INFORMATION FOR SEQ ID N0:27:
(i) SEQUENCB CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUBNCE DESCRIPTION: SEQ ID N0:27:
GACAAGAATC CTCACAATAC CRCAGArTCT 30 (2)INFORMATION FOR SEQ ID N0:28:
WO 93/13120 ~ 12 ~ 9 2 8 PCT/US92/I 1 t65 (i> SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:28:
(2) INFORMATION FOR SEQ ID N0:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single ' (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:29:
CACCATATTC TTGGGAAC)1A GAKCTACAGC 30 (2) INFORMATION FOR SEQ ID N0:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDSDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:30:
ACACTTCCGG ARAC1'ACfGT' TGTTAGACGA 30 (2) INFORMATION FOR SEQ ID N0:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:31:
GTVI'CITrYG GAGIGTGGAT TCGCACTCCS 30 WO 93/13120 ~ 12 ~ 9 2 8 PCT/US92/11165 (2) INFQRMATION FOR SEQ ID N0:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:32:
T1'GGAGCWWC TGTG«AG1TA CTCTCKTTrT 30 (2) INFORMATION FOR SEQ ID N0:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQU~NCB DESCRIPTION: SEQ ID N0:33:
(2) INFORMATION FOR SEQ ID N0:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:34:
AAWGRTCZTr GTAYTAGGAG GClGTAGGC~1 30 (2) INFORMATION FOR SEQ ID N0:35:
(i1 SEQUENCB CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ~12~928 WO 93/13120 PCf/US92/11165 _37_ (xi) SEQUENCE DESCRIPTION: SEQ ID N0:35:
RGAC'IGGC''AG GAGYTGGC,GG AGGAGATTAG
(2) INFORMATION FOR SEQ ID N0:36:
5 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear 10 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:36:
CCTTGAGrCM TACTT'CJIpT~G A~,T~.~
(2) INFORMATION FOR SEQ ID N0:37: w (i) SEQUENCE CHARACTERISTICS:
15 (A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCB DESCRIPTION: SEQ ID N0:37:
cTCrcTCCCr TcrcaTCrcc cccwcccn;T
(2) INFORMATION FOR SEQ ID N0:3B:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid 25 (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCB DESCRIPTION: SEQ ID N0:38:
3 0 ~ T~~G csteccTCrcc (2) INFORMATION FOR SEQ ID N0:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRAND&DNfiSS: single 35 (D) TOPOLOGY: linear WO 93/13120 PCr/US92/11165 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:39:
GGCTCSTCfG CCGATCCATA CTGCGGAACT ~ 30.
(2) INFORMATION FOR SEQ ID N0:40:
(I) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:40:
(2) INFORMATION FOR SEQ ID N0:41:
(I) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SSQ ID N0:41:
GTGGCTCCAG TTC29'.,GAACA GTAAACCCTG 30 (2) INFORMATION FOR SEQ ID N0:42:
(I) SEQUENCB CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:42:
KAARCAGGCT TTYAC1TIY.T CGCCAACTTA 30 (2) INFORMATION FOR SEQ ID N0:43:
(I) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs 3S (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:43:
CCTCCKCCTG CCtCYACCAA TCGSCAGTCA 30 (2) INFORMATION FOR SEQ ID N0:44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:44:
ACCAATTTTC T1'YTGIrTYT GGGTATACAT 30 (2) INFORMATION FOR SEQ ID N0:45:
(i) SEQUfiNCB CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:45:
TATTCCCATC CCATCRTCCT GGGC1'P1'CGS 30 (2) INFORMATION FOR SEQ ID N0:46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:46:
(2)INFORMATION FOR SEQ ID N0:47:
WO 93/13120 ~ ~ ~ ~ ~ ~ ~ PCT/US92/t 1165 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:47:
CGTAGGGCIT TCCCCCACfG TTTGGCI1TC 30 (2) INFORMATION FOR SEQ ID N0:48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single -(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:48:
GCTCAGTTTA CfAGIGCCAT TTGTTCAGTG 30 (2) INFORMATION FOR SEQ ID N0:49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:49:
CCTATGGGAG KGGGCCTCAG YCCGTTI'CTC 30 (2) INFORMATION FOR SEQ ID NO:50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:50:
GTCCCCTAGA AGAAGAACTC CCTCGCCfCG 30 ", WO 93/13120 ~ 1 2 ~ g 2 g PCT/US92/11165 (2) INFORMATION FOR SEQ ID N0:51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:51:
ACGMAGRTCT CMATCGCCGC C1'CGCAGAAG A 31 (2) INFORMATION FOR SEQ ID N0:52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:52:
(2) INFORMATION FOR SEQ ID N0:53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single, (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:53:
GACTCATAAG GTSGGRAACT TTACKGGGCZ' 30 (2) INFORMATION FOR SEQ ID N0:54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear WO 93/13120 12 4 ~ 2 g PCT/US92/1116s (xi) SEQUENCB DESCRIPTION: SEQ ID N0:54:
AGGCATAGGA CCCG1'GTCZT 20 (2) INFORMATION FOR SEQ ID NO:55:
(i1 SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear 10 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:55:
crrcrrrcca caraacTCCrc ?o WO 93/13120 ? 1 2 L~ 9 2 ~ PCT/US92/11165 FIL°_ NP11E LAST ACCE66 OAT~ ~..~EFTEC cILE NAHE ~9T ACCES- PATE
C2cRTrC
FILE TY°E~ 9YNTHE6I5 CYCL
%SC-~ 08 Z7. 199108 27r.1991 o.a%9-S 08 ZT, 199i08 '_'.laal 6. "
I.Z%D-n08 27. 199108 27,1991 1.2%-0 08 27. 199108 Z7.1991 aseeef30t 07, 19900t 07,1990 eeaf3 0t 07, 19900t 07,1990 lOeeeP321 07, 199001 07,1998 naaf3 0i 07, 199001 07,1990 l0haaf30t 07, 199001 27.,1990 rnsaf3 01 07, 1990Bt 07,1990 t0rnaaf3-01 87, 199001 07,I99B saeef3 0I 07, 199001 07,1998 cef3 01 07, 199001 07,1998 t0eaf3 01 07, 199001 07,1990 l0hof3 81 07, 19900t 07,i990 rnsf3 01 07. 19900t 07.1990 t0rnaf301 07, 199001 07,1990 aaeaaft 01 07, 19980f 07,1990 ceafl 0I 07, 19900I 07,1990 IOeeafl 01 07, 199001 07,1990 noaft 0t 07, 199001 07,1990 t0hoafl 0t 07, 199001 07,1990 rneafl 01 07. 199001 07,1990 l0rnaafl 01 87, 199001 07,1990 asceft 01 07, 199001 07,1990 esfl 01 A7, 199001 07,1990 l0ceft 0t 07, 199001 07,1990 l0hofl 0t 07, 19900t 07.1990 rnalt 01 07, 1990AI 07,1990 l0rnefl 01 07, 199001 07,1990 FILE TYPE: BOTTLE CHAH6E PROCEDURE
be 18 07 01, 1986 07 01. 1986 be 17 07 01, 1986 07 01, 1986 tie i6 07 01, 1986 07 0t, 1986 be 15 07 8t, 1986 87 01, 1986 be to 07 01, 1986 07 01, 1986 be 13 07 81. 1986 87 01, 1986 be IZ 07 0t, 198fi 07 81, 1986 be 11 07 81, 1986 07 01, 1986 be 10 07 01, 1986 07 01, 1986 be 9 07 01, 1986 07 01, 1966 he 8a 07 01. 1966 07 01. 1986 be 7 07 01. 1986 07 01, 1986 be a 07 01, 1986 07 01, 1986 be 5 87 01, 1986 07 01., 1986 tie a 07 01, 1986 07 0I, 1986 tic 3 87 01, 1986 07 0t, 1986 oc 2 07 01, 1986 07 0t~, 1986 be I 07 0I, 1986 07 Bt, 1986 FILE TYPE:. ENO PROCEDURE
CAP-PRIM '08 2T. 1991 08 27. 1991 CE NH3 88 Z7, 1991 88 27. 1991 deoree 10 88, 1990 18 88. 1990 deoreel0 10 08, 1990 t0 08, 1990 deorho 18 08, 1990 I0 08, 1998 deorhol0 10 0B, 1998 18 08, 1990 deorna t0 08, I99A 10 08. 1998 deprnal0 10 08. 1990 10 08, 1990 FILE TYPE: 8E6IH PROCEDURE .
STD PR~° 88 27, 1991 88 27. 1991 onos803 87 81, 1966 07 01, 1986 FILE TYPE: SHUT-0O~H PROCEDURE
clean083 87 01, 1986 A7 81, 1986 FILE TYPE: DNA SEOUEHCE9 I6%-. 88 Z7, 1991 08 27. 1991 _ IS%-I 08 27, 1991 0B 2'. 1991 WO 93/13120 2 1 2 4 9 2 8 P~/US92/11165 4 ~I
STEP FUNCTION STEP STEP ACTIVE FOR 8R9E9 SrlFc N M . 3 LARNE T-.~F. R- 6 r 1 10 i19 To Ueate 3 Yes Yes Yes Yea Yea Yea Yes Yea ? 3 i16 To Column10 Yea Yes Yea Yea Yes Yea Yes Yea 3 Z Reverse Flusn~ Yea Yes Yes Yes Yea Yes Yes Yea s I Bloek Fluan 3 Yes Yes Yea Yea Yes Yes Yes Yea ~ Aovenee FC t Yea Yea Yea Yea Yes Yea Yes o' 29 . 3 Yea Yes ~ it5 Phoa Preo 1 Yea Yea Yea Yea Yea Yes Yea 6rouo I On Yea Yea Yea Yes Yea Yaa Yes Yea 8 98 TET To Coluwn1A Yea Yes Yea Yes Yes Yea Yes Yaa 9 19 6aTET To Col 6 Yea Yes Yes Yea Yes Yes Yes I Yea 9A TET To Columns Yes Yes Yes Yea Yea Yea Yes 11 -a6 6rouo 1 Off I Yes Yea Yes Yea Yea Yea Yes Yea Yea IZ .a7 6rouo 2 On 1 Yea Yea Yes Yes Yes Yes Yes Yea 13 90 TcT To Coluwn10 Yea Yea Yea Yea Yea Yaa Yea Yea is 28 9TET To Col 8 Yea Yea Yea Yes Yes Yea Yea 2 Yea 98 TET To Column' Yea Yea Yes Yea Yea Yea Yea Yea I6 -t8 6rouo 2 Off 1 Yea Yes Yes Yea Yea Yea Yea 17 it9 6rouo 3 On I Yea Yea Yes Yea Yea Yea Yea Yea Yea I6 98 TET To Coluwn18 Yea Yes Yaa Yea Yea Yea Yea 19 21 8TET To Col 8 Yea Yea 28 98 3 t Yes Yea Yea Yea Yea Yea Ye TET To Coluwn Yea Yea Yees Yea Yea Ysa Yea Yea a 21 -58 6rouo 3 Off 1 Yes Yea Yaa Yas Yea Yea Yes ZZ t Uett IS Yea Yes 23 ~S 6rouo I On 1 Yes Yea Yea Yaa Yes Yea Yea Zs 90 Tc~ To Coluwn18 Yea Yea Z5 i9 BTET To Col 6 Yes Yes Yea Yea Yes Yea Yes 26 90 1 4 Yna Yea Z7 -t6 TET To Column1 Yea Yes Yea Yea Yea Yea Yea 26 ~7 6rouo 1 Ott 1 Yes Yea 29 90 6rouo 2 On 10 Yea Yea Yea Yea Yes Yea Yea 38 ~ 7ET To Coluwn8 Yea Yes 31 Z8 BtTET To Col t Yea Yes Yea Yea Yea Yes Yes 32 9A Z I Yaa Yes 33 -t8~ TET To Column1 Yea Yes Yea Yea Yea Yea Yea 34 it9 Group 2 Otf 18 Tea Yes 35 9A 6rouo 3 On 8 Yes Yea Yea Yea Yea Yes Yea 36 21 TE7 To Coluwn1 t Yea Yea 37 98 BfTET To Cal t Yea Yea Yea Yas Yea Yea Yea 38 -58 3 ' 3B Yea Yes 39 t TET To Coluwn1 Yes Yes Yea Yaa Yea Yes Yea t8 X45 6rouo 3 Otf 10 Yea Yes t1 98 fait B Yea Yea Yea Yea Yea Yea Yea tZ 19 6rouo I On t Yea Yer s3 9A TET To Coluwnt Yaa Yes Yea Ysa Yea Yea Yea -tfi BtTET To Col Yea 1 Yas Yes Yaa Yea Yea Yea TET To Coluwn Yes 6rouo 1 Otf Yea Yea Yes Yaa Yea~YSa Yes Yea Yea Yea Yes Yea Yea Yes Yaa Yea Yea Yea Yea Yes Yee Yva Yea YeaYaa Yes Yes Yea Yas Yes Yea Yaa Yea Yea Yu Yea Yea Yes Yea Yea Yea Yas Yea Yes Yea Yes Yea Yea Yes Yea Yea Yea Yea Yea Yea Yes Yea Yea Yea Yea Yea Yea Yea Yea Yea Yes Yea Yes Yes Yea <Conttnueo next OaOe.>
WO 93/13120 ~ 12 4 9 2 8 PC1'/US92/11165 STEP FUNCTION STEP STEP ACTIVE FOR HR9E9 SAF=
NUhBEFt NH11~ T M 9 ~ ~ ' -' S--n al X67 or9Up Z ~n t Yea Yea Yea Yea Yea Y.a Yea ~~a 6~ 90 TET To Column 10 Yea Yes Yea Yea Yea Yea Yes tes a6 20 B~TET To Col : 9 Yes Yes Yes Yea Yes Yea Yea taa a7 90 TET To CoLuwn a Yes Yea Yes Yes Yea Yes Yea iaa a8 -a8 Group 2 Off 1 Yes Yea Yea Yea Yes Yea Yea Y-_a d9 ~a9 6rouo 3 On t Yes Yea Yea Yea Yna Yea Yea Yea 90 TET To Coluwn 10 Yea Yea Yea Yea Yea Yea Yea res 51 . 21 6~TE7 To Col 3 ' B Yea Yea Yea Yea Yea Yea Yaa Yes 52 90 T-ET To Coluwn a Yaa Yea Yes Yea Yea Yea Yea Yea 53 -50 6rouo 3 Off t Yea Yea Yea Yes Yea Yes Yas Yes 5a a yslt 30 Yes Yea Yna Yea Yes Yes Yea Yea ~a5 orouo 1 On 1 Yea Yea Yes Yea Yea Yea Yea Yea S6 90 TET To Coluwn 10 Yea Yea Yea Yes Yea Yea Yea Yes 57 I9 HrTET to Col t 8 Yea Yes Yes Yea Yea Yea Yes Yes 58 90 TET To Coluwn a Yes Yes Yea Yea Yea Yes Yes Yaa 59 -a6 Group I Off I Yea Yaa Yea Yea Yea Yea Yes Yes ~a7 Group 2 On I Yea Yea Yea Yea Yea YeaYea Yea 6I 90 Tc7 To Column 10 Yes Yea Yaa Yes Yea Yea Yea Yea 6Z 20 HtTET To Col 2 8 Vas Yaa Yea Yar Yea Yea Yes Yea 63 9A TET To Coluwn 4 Yes Yes Yea Yea Yea Yes Yea Yea 6a -a8 Group 2 Off 1 Yea Yas Yea Yea Yaa Yaa Yea Yea ~a9 Group 3 On I Yea Yes Yea Yaa Yaa Yas Yea Yes 66 90 TET To Coluwn 1H Yea Yea Yes Yea Yea Yea Yes Yes 67 Z1 H~TET To Col 3 8 Yea Yes Yaa Yes Yaa Yes Yea Yea 66 9A TET To Colwn a Yea Yea Yea Yea Yea Yea Yea Yea 69 -58 Group 3 Off 1 Yea Yea Yea Yes Yas Yea Ysa YGs a ualt 30 Yea Yea Yea Yea Yea Yea Yea Yes 7t ~a5 Group I On I Ysa Yes Yea Yea Yea Yea Yea rea 72 90 TET To Coluwn 10 Yes Yea Yaa Yea Yea Yea Yea Yea 73 19 BtTET To Col 1 8 Yea Yea Yea Yea Yes Yea Yea Yea 7a 9A TET To Colwn a Yaa Yaa Yea Yea Yea Yes Yes Yes -a6 Group 1 Off I Yes Yea Yea Yaa Yea Yea Yes Yes 76 '~a7 firouo 2 On 1 Yss Ysa Yes Ysa Yea Yaa Yaa Yes 77 9i TET To Colwn 10 Yea Yaa Yas Yes Yes Yea Yea . Yes 7H 2~ B~TET To Col 2 8 Yas Yes Yea Yaa Yea Yaa Yea Yea 79 9i TET To Colwn a Vas Yea Yes Ysa Yea Yaa Yea Yea HA -a8 Group Z Off I Yes Yea Yea Yea Yes Yes Yes Yes Bt ~a9 6reup 3 On I Yea Yea Yea Yea Yes Yaa Yea Yes H2 9H TET To Coluwn ~ 10 Yss Yea Yes Yea Yea Yea Yes Yea 63 21 HaTET To Col 3 H Yea Yea Yee Yea Yaa Yea Yes Yea H4 90 TET Ta Colwn a Yea Yea Yea Yea Yea Yea Yea Yes HS -5B Group 3 Off I Yea Yea Yaa Yea Yea Yas Yes Yea H6 a yell 3H Yes Yes Yas Yas Yea Yes Yaa Yea H7 ia5 Group I On 1 Yaa Yaa Yaa Yea Yaa Yea Yns Yea 88 9H TET 7o Colwn I0 Yes Yes Yaa Vas Yea Yea Yes Yea_ (Coneinued neat ps0e->
WO 93/13120 ~ 2 ~ 9 2 8 PCT/US92/1 I 165 9TE? FUNCTION 9TE. gTE? ACTIVE FOfY 8H9E9 SAFc N h x NtIwE TIME 9 r r E '~ c-.~~
g9 19 GiET To Col _ 0 Yas Yes Yea Yes Yes Yes Yes I Yes 9p 9B iET To Column .t Yes Yes Yea Yes Yea Yes Yes Yes 91 -La 6rou0 1 Off I Yea Yes Yes Yea Yea Yes Y-_s Yes g2 +a7 Grouo 2 On t Yea Yea Yes Yes Tea Yea Yes Yes 93 90 TET To Coluwn 1B Yes Yea Yes Yes Yes Yea Yes Yes 9a Z8 B+TET To CoL 8 Yes Yea Yes Yea Yea Yes Yes 2 Yes 95 98 TET To Coluwn a Yes Yea Yes Yea Yea Yea Yes ~ Yea 96 ~ 6rou0 2 Oft 1 Yea Yea Yea Yea Yes Yes Yes -a8 Yes g7 +a9 Group 3 On I Yea Yes Yea Yas Yaa Yes Yes Yes 9A 90 TcT To Coluwn I8 Yea Yes Yes Yea Yes Yes Yes Yes 99 2i B+TET To Co1 B Yea Yea Yea Yes Yes Yea Yea 3 Yes 180 98 T'cT To Coluwna Yaa Yes Yes Yaa Yes Yea Yes Yea I81 -tea Group 3 Oft I Yea Yes Yea Yes Yes Yea Yea Yea 182 a Wait 30 Yes Yea Yea Yea Yea Yes Yes Yea 103 a6 6rouo I On 1 Yna Yea Yes Yea Yea Yea Yes Yea 10a 98 TET To Coluwn 10 Yea Yes Yea Yea Yes Yes Yes Yea 105 19 B+TET To Col 8 Yea Yea Yaa Yea Yaa Yea Yes I Yea 186 98 TcT To Coluwn Yea Yea Yea Yea Yes Yes Yea Yea 107 -a6 Group 1 Otf I Yea Yea Yaa Yea Yaa Yea Yes Yea 188 +a7 Group 2 On I Yaa Yea Yaa Yea Yas Yea Yea Yea 189 9A TET To Coluwn 18 Ysa Yaa Yaa Yaa Yaa Yes Yea Yaa II0 28 8+TET To Col 8 Yas Yaa Yea Yea Yaa Yas Yea 2 Yaa 111 90 TET To Coluwn t Yes Yes Yea Yes Yea Yea Yea Yea 112 -t8 Group 2 Off 1 Yes Yes Yea Yea Yea Yes Yes Yea 113 +a9 Group 3 On 1 Yea Yaa Yas Yea Yes Yea Yes Yes 4 98 wn 18 Yaa Yea Yea Ysa Yea Yes Yea TET To Col Yea 11 21 u A Yea Yas Yea Yea Yaa Yea Yea I15 6+TET To Col Yea 116 98 TET To Coluwn a Yes Yea Yaa Yes Yea Ysa Yea Yea 117 -Sa Group 3 Oft I Vas Yea Yea Yea Yea Ysa Yea Yea 118 a hut 38 Ysa Yaa Yea Yea Yaa Yea Yes Yes 1i9 +a5 Group I On 1 Yea Yes Yea Yaa Yes Yea Yes Yes 120 90 TET To Coluwn IA Yaa Yes Yea Yaa Yea Yes Yes Yea I21 ' B+TET To Col A Yes Yas Yes Yaa Yaa Yes Yea 19 1 Yaa 122 9i TET To Cpluwn L Yea Yes Yaa Yas Yea Yea Yes Yes 123 -I6 Group I Off I Yea Yes Yas Yaa Yes Yea Yea Yaa I2a +a7 6rouo 2 On 1 Yes Yas Yea Yaa Yes Yea Yes Yaa 125 9A TET~ To ColuwnIA Yea Yea Yea Yes Yea Yes Yea Yea I26 2i 8+TET To Col 8 Vas Yaa Yea Yea Yaa Yes Yes 2 Yaa 127 98 TcT To Coluwn ~ a Yea Yea Yea Yea Yea Yes Yea Yea 128 -a8 Group 2 Off 1 Yaa Yaa Yea Yu Yas Yaa Yes Yea 129 a9 Group 3 On I Yea Yae Yee Yae Yea Yea Yes Yea 138 9A TET To Caluwn 18 Yea Yea Yaa Yas Yea Yea Yes Yas 131 2I B+TT To Col 8 Yea Yes Yes Yea Yes Yea Yea 3 Yes 132 98 TET To Coluwn 4 Yaa Yes Yes Yes Yaa Yea Yns Yes 133 -58 6rouo 3 Off I Yes Yaa Yea Yea Yaa Yes Yes Yes _ (Conttnueo next oepa.l .... W093/13120 ~ pCT/US92/Itl6;
STE? FUNCTION STEP STEP HCTIVE
iAFc FAR
BHSEi N h s NllnE TIhE R 5 y > > ' iT~
i3d 1 :lest ~0 Yes YeaYeaYesYesYeaYes Yes 135 id 318 To Uaate i Yes YesYesYesYesYeaYts Yea 136 . Reverse Fluah~ Yes YeaYeaYeaYesYeaYes Yes 137 1 8lotk Flush d Yea YesYeaYeaYeaYeaYes Yea 138 31 SIS To usate 3 Yea YesYesYeaYeaYeaYes Yea 139 13 f15 To Column22 Yea YeaYeaYesYesYesYea Yes 1a0 10 f18 To 4laateS Yea YeaYeaYeaYeaYeaYea Yea 1t1 d ~ett 30 Yea YeaYeaYaaYesYeaYea Yea It2 2 Reverse Fluah6 Yea YeaYeaYeaYeaYeaYea Vas 1t3 I Block Fluah Yes YeaYesYeaYesYeaYea Yea tat ? fIB To Column10 Yes YeaYeaYeaYeaYeaYea Yea 115 3d FLuah to Waate~ Yea YeaYeaYesYesYesYea Yea I46 3 318 To Column10 Yea YesYeaYeaYesYesYea Yea 1a7 . Reverse Fluan5 Yea YeaYesYeaYeaYeaYea Yes It8 9 a18 To Columnf0 Yea YeaYesYeaYeaYeaYes Yes It9 2 Reverse FlusnS Yea YeaYeaYeaYeaYesYea Yes I50 9 f18 To Column10 Yea YeaYesYeaYesYesYea Yea 151 2 Reverse Fluah5 Yea YeaYesYeaYeaYesYes Yea 152 1 Hloek flush t Yes YesYesYeaYasYesYes Yes 153 33 Cyela Entry t Yas YeaYesYssYesYesYea Yen 154 5 4laate-Port I Yas YaaYsaYeaYeaYeaYea Yea 155 37 Ralay 3 PulseI Yaa YeaYeaYesYeaYeaYsa Yes 156 82 ilt To ~aate 3 Yea YaaYesYeaYeaYesYea Yea 157 30 3l7 To haste 3 Yea YeaYeaYeaYeaYeaYea Yea I58 10 f18 To yaste S Yea YeaYesYeaYeaYeaYaa Yea 159 9 t18 To Column20 Yea YeaYeaYesYeaYesYea Yes 160 II :l7 To Column60 Yea YeaYesYeaYesYeaYes No 161 1d fIL 7o Column20 Yea YesYeaYeaYeaYeaYea No 162 2 Reerse Fluah 7 Yes YesYeaYesYesYeaYea No 163 11 t17 To ColumnIS Yea YeaYeaYeaYsaYeaYea No I6t 3t Fluah to haste-5 Yes YaaYeaYeaYesYeaYea No I65 11 ill To ColumnIS Yes YeaYeaYeaYeaYesYes No I66 ' Rweraa Flush 5 Yea YeaYeaYeaYeaYsaYes Ho l67 It ttt 7o Colwn 20 ~ YssYasYsaYeaYesYea No Yes 168 .3t Fluah to haste10 Yes YeaYasYesYeaYesYea No 169 7 4laate-BottlsI Yea YeaYesYsaYeaYeaYea Yes 170 9 f18 To Column10 Yea Yea Yes YsaYea Yea Yea Yea 171 2 Reveraa Flush5 Yes Yas Yea YasYes Yes Yes Vas 172 9 fib To Calunn~ 10 Yes Yes Yea Yes Yss Yea Yea Yea 173 Z Reveraa FlushS Yea Yea Yea Yea Yea Yes Yea Yaa 17t 9 i18 To Column10 Yes Yea Yea Yaa Yae Yas Yea Yea 175 2 Revsrae Fluah5 Yes Yes Yes Yes Yea Yea Yea Yes ' i76 1 0loek Fluah 3 Yea Yea Yes Yea Yea Yea Yea Yes WO 93/13120 ~ PCT/US92/11165 STEP FUNCTION STEP STEP RCTIV FOR BR9E9 SRF=
NUN9ER 3 TINE A ~ r T 5 'T 5~F' NRNF
I 10 S18 To ~aate 3 Yea Yes Yes Yes Yes Yea Vea Yea 2 9 a18 To Column10 Yes Yes Yea Yes Yes Yes Yaa Yea S 2 Reverse Fluan~ Yes Yea Yes Yea Yes Yea Y-_a a I Block Flush 3 Yea Yea 5 Advenee FC I Yes Yea Yea Yea Yes Yea Yes 6 ~ Phos Prep 3 Yea Yes ? 29 Group 1 On 1 Yea Yes Yea Yaa Yaa Yaa Yea 8 +a5 TET To ColumnI0 Yea Yea 9 90 B+TET To Col 8 Yea Yea Yea Yes Yes Yea Yes 19 1 s Yes Yea It 90 TcT To ColumnI Yea Yes Yea Yaa Yes Yes Yes -46 Group 1 Off Yea Yea Yea Yea Yaa Yea Yes Vaa Yes Yes Yea Yea Yea Yes Y'ea Yea Yea Yea Yaa Yea Yea Yes Yes Yea Yes Yea Yea Yes Yea 1= a7 Group Z On 1 Yea Yea Yes Yea Yea Yes Yea Yea 13 a0 TET To Coluan10 Yea Yea Yea Yea Yea Ysa Yea Is 29 BTcT To Cal 8 Yes Yes IS 90 Z < Yes Yes Yea Yea Tea Yea Yes 16 W8 TET To Column1 Yea Yea 17 a9 Group 2 Otf I Yea Yea Yea Yes Yea Yea - Yea 18 90 Group 3 On 10 Yea Yes t9 21 TET To Column8 Yea Yaa Yes Yea Yea Yea Yea 90 B~TET To Col a Yas Yea 21 -50 3 1 Yea Yea Yas Yea Yes Yes Yes t TET To ColuwnIS Yes Yes ~3 a5 Group 3 Oft 1 Yes Yea Yes Yea Yea Yea Yea 26 90 Matt 10 Yea Yes 19 6rouD 1 On 6 Yea Yes Yea Yea Yes Yea Yea 25 90 TET To Columna Yea Yea 27 -t6 B+TET To Col 1 Yea Yee Yea Yea Yes Yes Yea 2g sa7 1 I Yea Yea 29 90 TET To Column10 Yes Yea Yes Yea Yea Yea Yea 2A Group 1 Oft 8 Yea Yea 31 '9A Group 2 On L Yes Yea Yes Yaa Yes Yes Yea 32 -a8 TET To Coluwnt Yea Yes 33 a9 BaTET To Col I Yea Yea Yea Yea Yaa Yea Yea 3a 9A 2 10 Yes Yea Z1 TET To Column9 Yes Yea Yna Yea Yas Yaa Yea 36 9A Group 2 Off 4 Yea Yes 37 -58 Group 3 On t Yea Yes Yea Yaa Yea Yea Yes 38 TET To Column30 Yea Yes 39 +45 B~TET To Col 1 Yea Yea Yes Yes Yea Yes Yea a0 9A 3 IA Yea Yea at 19 TET To Coluwn8 Yea Yna Yea Yes Yea Yea Yea L2 9A ~6roup 3 Off a Yea Yea a3 -16 felt 1 Yes Yes Yaa Yea Yna Yea Yea Group t On Yea TET To Coluwn Yea Yes Yea Yea Yea Yaa B~TET To Cal Yes I Yea Yes Yea Yes Yes Yes TET To Coluwn Yea Group I Off Yes Yea Yea Yaa Yaa Yea Yea Yes Yaa Yea Yaa Yas Yea Yea Yea Yes Yea Yea Yea Yes Yea Yes Yaa Yes Yes Yas Yea Yes Yea Yea Yes Yea Yea Yea Yea Yes Yea Yea Yaa Yes Yea Yea Yea Yaa Ysa Ysa Yes Yea Yes Yea Yea Yea Yea YesYsa Yea Yes Yaa Yaa Yea Yea Yes Yas Yea Yea Yea Yea Yea Yes Yee Yes Yea Yea Yea Yea Yea Yna Yes Yes Yes Yaa Yea Yes Yas Yea Yes Yea Yas Yea Yes Yea tContinued next pape.) WO 93/13120 21 ~ ~ 9 2 8 PCT/US92/11165 STEP FUNCTION STEP STEP ACTIVE
SpF=
FOR
ARSES
NUMBER' 3 NHNE TiflE p _ _ -4i +d7 Sroup Z On - t Yea YeaYeaYeaYesYeaYes Y--_a 63 90 T=T To Calunn 10 Yea YesYeaYeaYeaYeaY-ssi_a Sd 20 B+TET To Col d Yea YeaYeaYesYtsYesYes Yca Z
i7 90 TET To Column d Yes YesYeaYeaYesYesYes Yea SA -dA 6reup 2 Off t Yea YeaYeaYeaYesYesYea Yea a9 +d9 6rouo 3 On I Yea YesYeaYeaYeaYeaYes Yea 60 90 TET To Column 10 Yes YeaYeaYeaYesYeaYea Yea 51 ~ B+TET To Col 8 Yea YesYeaYaaYesYeaYes Yes 21 3 ~
52 98 TET To Column d Yea YeaYeaYesYesYesYea Yea 53 -68 Group 3 Oft I Yaa YeaYasYeaYeaYeaYea Yes 5s a welt 30 Yea YeaYeaYeaYeaYeaYes Yea __ a5 Group I On I Yea YeaYeaYesYeaYeaYes Yes ~d 90 TET To Column 10 Vea YeaYeaYeaYeaYeaYea Yea ~7 13 6+TET To Col 3 Yea YesYeaYeaYesYeaYea Yes i ~d 90 TET To Column 6 Yea YeaYeaYaaYeaYeaYea Yes ~3 -a6 Group I Oft 1 Yea YeaYeaYeaYeaYeaYes Yes 60 +a7 Group .2 On 1 Yaa YesYeaYeaYeaYaaYes Yea 61 90 TET To Column I8 Yes YeaYeaYesYeaYeaYea Yes 62 20 B+TET To Col A Yea YesYasYaaYeaYeaYaa Yes 63 90 TET To Column a Yaa YeaYeaYaaYeaYeaYea Yes 61 -18 Group 2 Oft 1 Yes YeaYasYeaYaaYeaYea Yes 65 +a9 6rpup 3 On 1 Yaa YeaYesYasYesYeaYaa Yaa 66 90 T_T To Coluwn IB Yes YeaYasYeaYaa.YeaYes Vas 67 21 B+TET To Col B Yea YesYeaYaaYeaYesYea Yes 6A 98 TET To Coluwn a Yes YeaYeaYaaYeaYeaYea Yea 69 -SA Group 3 Otf I Yaa YeaYeaYesYasYaaYaa Yes 70 t felt 30 Yea YeaYeaYeaYeaYaaYea Yea 71 d6 Group I On 1 Yaa YaaYesYeaYeaYesYea Yas T_ 98 TcT To Column I8 Yea YeaYeaYeaYasYesYas Yes 73 19 B+TET Ta Col 8 Yea YeaYaaYesYesYasYaa Yes I
74 98 TET To Coluwn < Yaa YeaYeaYesYesYesYes Yas 75 -t6 Group 1 Oft 1 Yna YeaYeaYesYsaYeaYas Yes 76 '+a7 Group 2 On 1 Yea YeaYeaYsaYesYeaYea Yes 77 9A TET To Coluwn 10 Yas YeaYesYaaYaaYeaYes .Yea 78 2'D B~TET To Col B Yea YeaYeaYeaYaaYaaYea Yes 79 99 TET To Column t Yea YesYesYesYaaYeaYea Yes A8 -a8 Group 2 Off I Yas YaaYeaYaaYeaYesYaa Yea 81 ~ 6rouo 3 On t Yea YeaYaaYu YasYeaYea Yes +19 8Z 90 TET To Column ~ IA Yas YesYaaYeaYeaYea Yea Yaa -83 21 B~TET To Col B Yas YesYasYaaYea Yea 3. Yea Yea 8t 9A TET To Coluwn ' d Yea YeaYu YesYasYea Yes Yea BS -58 Group 3 Off I Ysa YeaYesYeaYeaYea Yea Yes 86 4 Hatt 30 Yea YesYeaYea Yes Yaa Yes Yea 87 15 Group t On 1 Yes YaaYesYaaYaa Yea Yaa Yea 88 90 TeT To Column 18 Yaa Yn>=
Yea Yea Yea Yea Yaa Yea (Continueo naa paqe.l WO 93/13120 212 4 9 2 8 PCT/US92/1116s iTEP FUHCTIOH STEP STEP RC1IVE , GRFc FOR
H M a NRHE T h R a ~ r 9 Ty~
99 !9 9+TET To Col - B Yea YesYeaYesYea Tes Yes 1 Yes 90 90 TET To Coluwn a Yea Y-_sYesYeaTes Yes Yes Yes 91 -ao Group I Off 1 Yes YesYeaYeaYea Yes Yes Yes 91 a7 Group Z On I Yea YesYesYeaYea Yes Yes Yea 93 9d TET To Coluwn 10 Yea YeaYeaYesYes Yea Yaa Yes 9a 20 6+TET To Col 8 Yea YeaYeaYeaYes Yea Yes Z Yea 95 90 TET To Coluwn a Yea YeaYesYeaYea Yea Yea Yea 95 ' Group 2 Otf 1 Yea YesYesYeaTea Vas Yes -a8 ~ Yea 97 +a9 6rouo 3 On 1 Yea YeaYesYeaYes Yea Yea Yea 99 9A TET To Coluwn 10 Yes YeaYeaYeaYes Yea Yea Yaa 99 21 8+TET To Col 8 Yea YeaYeaYeaYea Yes Yea 3 Yna 100 98 TET To Coluwn a Yea YeaYesYnaYaa Yea Yea Yes 101 -~d 6rou0 3 Off I Yea YeaYesYesYea Yaa Yes Yea 102 ~ Wait 30 Yas YeaYeaYeaYea Yes Yes Yea 103 +a5 Grpup t On 1 Yea YeaYesYeaYes Yes Yes Yea Ida 90 TET To Coluwn t0 Yes YeaYeaYeaYea Yea Yea Yes 105 19 6+TET To Col 6 Yea YeaYeaYeaYea Yna Yes I Yea 106 9A TET To Column 4 Yes YeaYeaYaaYna Yes Yes Yna 107 -a6 Group 1 Off I Yes YeaYeaYeaYna Yea Yes Yea 188 +a7 Group 2 On I Yaa YeaYeaYeaYea Yes Yaa Yaa 109 98 TeT To Coluwn 18 Yss YeaYeaYeaYea Yas Yes Yea 118 20 B+TET To Col 6 Yas YeaYesYeaYea Yes Yea 2 Yea 111 90 TET To Coluwn a' Yea YeaYesYeaYea Ysa Yas Yea I11 -aA Group 2 Off I Yaa YasYesYaaYea Yas Yes Yea 113 +a9 6rpup 3 On 1 Yea YesYeaYesYes Yea Yea Yea Ila 90 TET To Caluwn i0 Yaa YeaYeaYaaYna Yea Yes Yea 115 21 B+TET To Col B Yea YeaYesYeaYea Yea Yea 3 Yea 116 90 TET To Coluwn 4 Yes YesYeaYeaYaa Yea Yes Yaa 117 -~d Group 3 Off 1 Yes YesYeaYesYea Yea Yea Yes 118 a halt 3A Yne YaaYeaYasYes Yes Yea Yes I19 +a5 Group 1 On 1 Yea YeaYeaYaaYes Yaa Yea Yns 110 9A TET To Coluwn IA Ysa YeaYaaYesYns Yaa Yes Yea 121 ' B+TET To Col H Yas YeaYaaYeaYea Yes Yes 19 1 Yea 1ZZ 9i TET To Coluwn a Yea YnsYeaYeaYea Yaa Yas Yea 123 -a6 Group 1 Off I Yna YnaYnsYesYea Yas Yes Yea 126 +a7 Group Z On 1 Yes Yaa Yea Yes Yes Yns Yaa Yna IZS 90 TET To Coluwn 18 Yes Yna Yea.
Yaa Yes Yaa Yas Yaa 125 20 B+TET To Col H Yaa Yea 2 Yes Yea Yea Yna Yea Yea 127 9A TET To Coluwn ~ a Yna Yea Yes Yea Yea Yea Yaa Yea 12B -18 Group Z Off 1 Yea Yea Yaa Yea Yna Ysa Ysa Yes 129 +a9 Group 3 On t Yna Yea Ynn Yes Yea Yns Yes Yas 13A 9A TET To Coluwn IA Yna Yes Yaa Yaa Yea Yna Yas Yes 131 21 8+TET To Cal 8 Yea Yna 3 Yea Yea Yea Yes Yes Ysa 132 9A TET To Coluwn a Yas Yes Yes Yes Yes Yes Yas Yea 133 -5d 6reup 3 Off 1 Yas s a a Yes s YeL
Ye Ye Ye Ye Yes (Continuep neat peqe.l WO 93/13120 ~ ~ ~ ~ ~ ~ PCT/US92/11165 5TE? FUNCTION STEP STEP ACTIVE FOR 8R9E6 SAFE
.
N M s NRNE TIhE q r ' ~ 95i-??
~
134 1 Uett ~0 Yes Yea Yes Yep Yea Yes Yes Yes ~
Ij~ is Cao Preo 3 Yea Yes Yes Yea Yea Yea Yes Yes 136 10 318 To ~este 3 Yea Yes Yea Yes Yes Yes Y--_a Yea 137 ~ Reverse Fluan ~ Yes Yes Yes Yea Yes Yea Yes Yes 136 I Block Ftush d Yea Yes Yes Yea Yes Yea Yes Yea 139 91 Ceo To Coluwn 2: Yep Yes Yes Yea Yes Yea Yes Yes 140 10 i18To ~eate 3- Yea Yea Yes Yea Ysa Yea Yes Yea 141 4 IJeit 30 Yea Yes Yea Yea Yea Yea Yea Yes 142 ~ Reverse Flush S Yea Yes Yes Yea Yea Yea Y--_s Yaa 143 1 Block Flush 4 Yea Yea Yea Yes Yea Yea Yna Yes 144 61 SIS To ~eate 3 Yea Yea Yes Yea Yea Yea Yea Yes 145 13 s15 To Column ZZ Yea Yea Yes Yea Yea Yea Yea Yes 146 10 =I8 To ~aate ~ Yes Yea Yes Yea Yea Yea Yea Yea 157 4 deft 3A Yea Yea Yes Yes Yes Yea Yes Yea 1t6 2 Reverse Flusn ~ Yea Yea Yes Yea Yes Yea Yes Yes 149 I Block Flush 4 Yea Yea Yea Yea Yes Yea Yes Yes 150 9 s18 To Coluwn 10 Yea Yea Yes Yes Yaa Yea Yea Yea 151 34 FLuah to 4laateS Yes Yea Yes Yes Yns Yea Yea Yea 152 9 s18 To Coluwn 10 Yea Yes Yea Yes Yea Yea Yea Yaa 153 2 Reverse Flush S Yaa Yes Yea Yea Yea Yea Yes Yea 154 9 tIB To Coluwn 10 Yea Yea Yea Yes Yea Yea Yes Yes 155 Z Reverse Flush S Yes Yaa Yes Yaa Yea Yea Yea Yaa 9 S18 To Coluwn 10 Yea Yea Yea Yes Yes Yes Yes Yea 156 2 Reverse Flush 5 Yea Yea Yea Yes Yea Yea Yea 157 Yea 158 I Btoek Flush 4 Yes Yea Yea Yes Yea Yea Yea Yea 159 33 Cyela Elltry 1 Yas Yea Yaa Yes Yas Yes Yea Yes 160 6 ~este-?ort 1 Yea Yes Yea Yes Yes Yea Yes Yes 161 37 Relay 3 Pulse 1 Yea Yea Yea Yea Yea Yea Yes Yea 162 82 t14 To 4leate 3 Yes Yes Yea Yea Yna Yea Yes Yea 38 f17 To Yeate 3 Yea Yes Yes Yea Yes Yea Yea Yes 163 H S Yea Yea Yes Yes Yaa Yas Yea t Yea T
164 I0 ea e o i18 9 i18 To Coluwn 20 Yes Yes Yes Yes Yes Yea Yea Yea 165 ~ f17 To Coluwn 60 Yea Yea Yes Yea Yea Yea No 11 Yas 166 14 f11 To Coluwn 20 Yea Yes Yes Yes Yea Yes No 167 Yea 168 2 Ravaraa Flush 7 Yes Yea Yea Yea Yea Yea No Yea Coluwn l5 Yns Yea Yes Yes Yes Yea No t17 T Yea 169 11 o S Yea Yea Yes Yes Yea Yea No 170 34 Flush to W Yes ate 171 11 :17 To Coluwn IS Yea Yes Yaa Yaa Yea Yes wo ~ Yas 1n Z Revarae Flush S Yaa Yea Yaa Yaa Yas Yea Ho Yea 173 1a flt To Coluwn 28 Yaa Yes Yea Yes Yes Yea No Yea 174 34 Flush to Wate 10 Yes Yea Yes Yea Yea Yea No Yea 175 7 4eate-9ottle 1 Yes Yes Yaa Yas Yes Yes Yea Yea 176 9 a18 To Coluwn 10 Yes Yes Yes Yea Yes Yes Yea Yea sh 5 Yea Yes Yes Yea Yea Yea Yes Fl Yea 177 Z u 10 Yea Yea Yes Yea Yea Yea Yea Reverse Yea -178 9 s18 To Coluwn (Continued neat papn.>
5TE? FUNCTION STEP STEP RCTIVE FOR BRSES 9RFc N M Ft HRHE TIFIE A r [ r ~ ''7 STy~
179 '_ aeverae Flush _ ~ Yea Yes Yea Yes Yea Yes Yea Yes 180 ? s18 io Colunn 10 Yes Yes Yea Yes Yes Yes Yes Yes 191 2 Reverse Flusn S Yes Tea Yes Yes Yes Yes Yea Yes 182 I Bloek Fluan 3 Yes Yea Yea Yes Yea 'tea Yes Yes 2.24928 STEP' FUNCTION STEP iTP ACTIVE FOR BRfii S:,F_ HUhBER . T h R C C -x NRHE
I 10 318 To IJeate2 Yea Yes Yes Yea Yes Yea Yes Yea 3 =I8 Ta Column9 Yes Yea Yea Yea Yea Yes Yes 3 _ r7e~erae Fluani Yes Yea s I Block Fluah 3 Yea Yes Yea Yea Yea Yes Yes > > Apvanee FC 1 Yes Yes 6 - 2B Phca Prep 3 Yea Yes Yes Yes Yea Yea Yes 7 1S Group I On I Yea Yes 8 90 TT To Column 6 Yea Yea Yea Yea Yea Yea Yes 9 19 BTET To Col 6 Yes Yes 90 I 3 Yea Yea Yes Yea Yes Yea Yes II 1_ TET To Column~ Yea Yes I= 90 BTET To Col 3 Yes Yea Yea Yea Yes Yes Yes 13 19 I 3 Yea Yea I.t 3 TET To Column1 Yes Yea Yea Yaa Yea Yes Yea Ii -a6 9TET To Col 1 Yea Yes i6 47 t I Yea Yea Yea Yea Yea Yea Yea 17 10 s18 To Column6 Yes Y-es 18 I Group 1 Ott 3 Yes Yes Yes Yea Yea Yea Yea 19 98 Group 2 On 6 Yea Yea 28 20 s18 To ~aate 6 Yea Yes Yes Yea Yes Yes Yes 21 90 Block Flush 3 Yes Yes.
22 20 TET To Coluwn3 Yea Yes Yea Yea Yea Yes Yea 23 90 BtTET To Col 3 Yea Yes 2a 20 2 3 Yea Yes Yea Yea rea res Yea 25 9 TET To ColumnI Yea Yes 25 -a8 B~TET To Col 1 Yea Yes Yea Yes Yea Yea Yea 27 a9 Z 1 Yea Yea 28 10 Tai To ColumnL Yea Yea Yea Yea Yea Yea Yes 29 I 8flET To Col 3 Yea Yea 3B 90 2 6 Yea Yea Yea Yea Yea Yea Yes 31 ' s18 To Column6 Yaa Yea 32 21 Group 2 Otf 3 Yea Yea Yes Yes Yea Yea Yes 33 9A 'vroup 3 On 3 Yea Yea 3t 21 s18 To ~aate 3 Yes Yea Yes Yea Yea Yea Yea 35 9A Bloek Fluah 3 Vas Yea 36 2I TET To Coluwn1 Yes Yea Yea Yaa Yea Yea Yes 37 9 BaTET To Col ~ 1 Yes Yes 38 -Sa 3 20 Yes Yea Yea Yea Yes Ysa Yes 39 t TET To ColuwnS Yea 48 2 BtTET To Col 2 Yea Yaa Yea Yes Yaa Yea Yes al 10 3 9 Yea Yea 42 9 TET To ColumnS Yea Yes Yea Yea Yea Yaa Yea a3 2 9;TET To Col 3 Yaa Yes_ 10 3 Yea Yes Yea Yes Yes Yaa f18 To Coluwn Yea 6reup 3 Oft Yea Yes Yes Yea Yea Yes IJait Yea Reverse Fluah Yaa Yea Yea Yea Yea Yes s1A To Yaste Yes sib To Column Yaa Yea Yea Yaa Yea Yas Reverae Flusn Ysa s18 To ~aate Yea Yes.Yes Yea Yea Yea Yea Yea Yas Yes Yea Yea Yea Yea Yes Yaa Yaa Yes Yaa Yea Yea Yes Yes Yea Yea Yes Yea Yna Ysa Ysa Yes Yes Yea Yea Yea Yes Yea Yea Yea Yea Yes Yea Yea Yea Yes Yea Yea Yea Yes Yes Yea Yes Yaa Yea Yea Yea Yea Yea Yea Yaa Yea Yea Yea Yea Yea Yes Yu Yea Yas Yes Yes Yea Yea Yea Yas Yea Yea Yea Yea Yea Yea Yes Yes Yea Yea Yes Yes Yea Yee (Continued next pepe.l WO 93/13120 ~ ~ PCT/US92/11165 STEP FUNCTION STEP STEP FOR9RSE5 SRF=
RCTIV ~
NR11E T h p [ ~ SS Tj r ' t4 . 9loek Fluah - 3 Y -d~ 15 Group 1 On ~ Yes _ Y a a6 90 TET To Column o Yes Yes t7 19 9+TET To Col a Yes Yea 68 90 TET To Column 3 Yea Yes a9 t9 8+TET To Col 3 Yes Yea 50 9A TET To Column 3 Yes Yes 51 - 19 9+TET To Col 3 Yea Yea t ~
52 9 S18 To Column I Yea Yes 53 -t6 Group l Off I Yea Yea 54 +t7 Group 2 On I Yes Yes ' SIB To ~aate 6 Yea Yea 3 Yes Yes 56 1 6loek Fluah 57 90 TET To Column o Yea Yes 59 20 9+TET To Col 6 Yea Yes .3 90 TET To Colwn 3 Yes Yea 60 2A B+TET To Col 3 Yes Yes 61 90 TET To Coluwn 3 Yea Yes -e2 Z8 B+TET To Col 3 Yea Yes Yea Yea 63 9 S18 To Column 1 , Yea Ye 6s -t8 Group 2 Ott 1 a Yea Yea 65 +t9 Group 3 On 1 Yea Yes 66 18 S18 To haste t 3 Yes Yes 67 1 6loek Fluah Yes Yea 66 98 TET To Column 6 69 21 8+TET To Col 6 Yaa Yes Yes Yea 78 90 TET To Column 3 Yea 71 21 9+TET To Col 3 Yes 3 Yea Yes 72 98 TET To Colwn Yea 73 21 9+TE7 To Col 3 Yea Yea Yea 74 9 SIB 7o Column 1 Yea 75 '-SA Group 3 Ott I Yes Yes 28 Yes 76 l Wdt 3 Yea YeaYeaTeaYea Yes Yes Yea 77 16 Cap Prep 5 Yea YeaYeaYeaYea Yea Yes Yea 76 Z Reverse Flush Y YaaYea Yes Yea a Yes 79 1 HloeE Flush 3 Yaa Yeae I2 Yea YesVasYaaYes Yes Yas Yea 80 91 Cao To Colwn ~ Y Yea Yea 91 I0 i18 To haste 3 Yaa VasYesYeaea Yes 9 Yea YaaYeaYesYaa Yea Yes Yea 92 t halt ' S Yes YesYesYesYea Yae Yes Yea 93 Z Revarae Flush Y YeaYeaVas Yea Yes Yea 8t 81 i15 To liaate 3 Yea ea Yes 1A Yea YesYesYaaYea Yee Yes 95 13 S15 To Colwn a YeaYea Yes Yes Y Yea 96 t8 i18 To llsate 3 Yes Yeae 15 Yea YeaYeaYeaYea Yea Yes_ Yea A7 t ~eit ~ Yea YeaYeaYesYea Yea Yes Yes 88 Z Reverse Flush (Continueo next peqe.>
", WO 93/13120 2 1 2 4 9 2 $ pCl~L7S92/11165 STEP FUNCTION STEP STEP RCTIVE
SPF'c FOR
BRfiES
N h c s NPnE T M a 6 G 5 ST~o 0g ? SIB To Column - 9 Yna YesYeaYesYeaYesYns Yea ?0 3l Fiush to UeateS Yes YeaYesYesYesYeaYes Yes 91 ? 318 To Column 9 Yes YesYeaYesYesYeaYes Yea 92 2 Reverse Fluan 5 Yes YeaYesYeaYeaYeaYea Yes 93 3 118 To Column 9 Yea YeaYesYesYesYeaYea Yes 9a ~ Reverse Flusn 5 Yea YeaYeaYesYeaYeaYea Yea g5 I 3loek Flush 3 Yea YesYeaYesYnaYesYea Yes 96 33 Cyele Erttry I Yea YeaYeaYasYeaYeaYaa Yes 97 9 1t8 Ta Column S Yaa YeaYeaYeaYeaYesYea Yes 9g 2 Reverse Fluan S Yea YesYeaYeaYesYesYes Yea 5 ~eate-Port I Yea YesYeaYeaYeaYeaYea Yes _ 30 317 To ~eate 3 Yes YeaYeaYeaYeaYeaYea Yea 101 II t17 To Column 7 Yea YeaYeaYeaYesYeaYes No 102 3a FLuah to 4laate1 Yea YeaYesYesYeaYesYea No 103 11 t17 To Column 7 Yea YeaYea-YSaYesYesYea No 10a 3a FLuah to ~aateI Yea YesYeaYesYesYeaYes No 105 It t17 To C0lunn 7 Yaa YnaYeaYeaYeaYeaYns No 106 36 Flush to ~eateI Yaa YeaYeaYesYeaYeaYaa No 107 11 a17 To Column 7 Yas YesYeaYeaYeaYeaYea No 10B 3a Flush to hastet Yea YaaYeaYeaYaaYeaYas No 109 11 617 To Coluwn 7 Yas YeaYeaYaaYeaYesYea No 110 34 Flush to lute 1 Yea YnaYasYasYeaYaaYea No 111 11 t17 To Column 7 Yaa YeaYesYaaYaaYeaYna No 112 3a Flush to ~aate~S Yas YeaYesYaaYesYeaYaa No 113 9 318 To Column 9 Yea YeaYeaYaaYaaYaaYea No 116 3a Flush to Waste7 Yes YesYesYeaYesYeaYea No 115 7 Lleate-Bottle 1 Yea YeaYnaYeaYeaYeaYea Yes 116 9 t18 To Colwn 9 Yea YeaYesYeaYeaYesYea Yes t17 2 Reverse Flush S Yea YesYeaYeaYeaYesYes Yes IIB 9 118 To Column 9 Yea YeaYesYesYeaYeaYns Yes 119 2 Reverse Fluan S Yes YeaYesYeaYeaYeaYea Yea 120 1 Bloek Flush 3 Yea YeaYeaYeaYeaYesYea Yes W093/13120 ~ PCh/US92/II165 STEP FUNCTION STEP STEP RCTIVE FOR gR6E6 9RFE
N t1 3 NRHE ~ R ' ' STe?
I 10 i18 To ueste = Yes Yea Yes Yss Yes Yes Yes fes 2 9 SIB To Column9 Yes Yea Yea Yea Yes Yea Y._s Yes 3 Z Reverse Flusn~ Yea Yea Yes Yes Yes Yes Yes Yes a I glopk Flush 3 Yea Yes Yea Yea Yes Yea fes Yes 5 ACvenpe FC 1 Yea Yea Yea Yea Yes Yea Yes Yea 2B Phos Prep 3 Yes Yes Yea Yea Yea Yes Yea Yea 7 +a5 6rouo I On I Yea Yna Yes Yea Yes Yea Yes Yea 8 90 TET To Column6 Yea Yea Yaa Yaa Yaa Yea Yes Yea 9 19 8+TET To Cal 6 Yaa Yea Yes Yea Yea Yea.~YeaYes I
90 TET To Coluwn3 Yes Yea Yea Yea Yes Yea Yes Yaa II 19 8+TET To Col 3 Yes Yea Yes Yea Yea Yea Yea I Yea 13 90 T_T To Column3 Yea Yea Yea Yes Yea Yea Yes Yea 13 19 B+TET To Col 3 Yea Yea Yea Yes Ysa Yea Yes 1 Yes Ia 3 t18 To Coluwn1 Yea Yea Yea Yea Yea Yea Ye_s Yes -a6 firoup 1 Ott I Yea Yea Yaa Yea Yes Yes Yes Yea 16 +a7 Group 2 On 1 Yes Yea Yea Yes Yes Yea Yea Yaa 17 I0 SIB.To haste a Yea Yea Yea Yes Yea Yea Yes Yea IB 1 Bloek Flush 3 Yes Yes Yaa Yaa Yaa Yea Yaa Yes "" - 19 90 TET To Column6 Yea Yes Yea Yea Yaa Yea Yea Yea Z8 28 8+TE7 To Col 6 Yaa Yaa Yes Yaa Yea Yn Yea Z Yaa Z1 90 TET To Column3 Yes Yes Yea Yaa Yaa Yea Yes Yea 22 20 B+TET To Col 3 Yes Yea Yea Yea Ysa Yea Yes 2 Yes Z3 90 TET To Coluwn3 Yea Yes Yaa Yes Yes Yaa Yes Yes 2a 20 g+TET To Co1 3 Yes Yea Yea Yaa Yea Yea Yes 2 Yea 9 s18 To Coluwn1 Yes~Yea Yea Yaa Yea Yea Yea Yes Z6 -a8 Group 2 Ott 1 Yea Yea Yea Yes Yes Yea Yes Yea Z7 +a9 Group 3 On 1 Yea Yes Yea Yea Yea Yes Yes Yea 28 10 SIB To Wstn 4 Yaa Yes Yea Yea Yea Yea Yes Yea 29 I Bloek Flush 3 Yea Yea Yea Yea Yea Yes Yea Yes 98 TET To Coluwn6 Yea Yss Yes Yes Yes Yea Yea Yes 31 ' B+TET To Col 6 Yes Yaa Yea Yes Yea Yea Yes 21 3 Yea 32 9A 7ET To Coluwn3 Yea Yea Yea Yaa Yae Yea . Yes Yea 33 21 g+TET To Col 3 Yes Yaa Yas Yea Yea Yea Yes 3 Yea 3a 9A TET To Coluwn3 Yas Yes Yes Yaa Yaa Yaa Yes Yea ' 21 B+TET 1o Col 3 Yes Yea Yea Yea Yea Yea Yea 35. 3 Yes . 9 :18 To Coluwn1 Yea Yea Yea Yu Yes Yea Yea 36 Yas 37 -50 firoup 3 Ott t Yes Yea Yes Yea Yaa Yea Yes Yea Z0 Yes Yes Yea Yas Yas Yes Yes Yes 3g a Halt 3 Yea Yes Yea Yaa Yaa Yaa Yes Yaa 39 16 Cap Prep sh S Yea Yea Yaa Yes Yaa Yea Ycs Fl Yea Z u 3 Yea Yea Yea Yes Yes Yea Yes ai t Reverse Yes ek Flueh Bl a2 91 o 12 Yes Yes Yea Ysa Yaa Yes Yea To Coluwn Yes C
ap 3 Yes Yes Yes Yea Yea Yea Yes Yea a3 10 fib To sate (Canttnueo next pa0a.>
.,. W093/13120 PCT/US92/11165 STEP FUNCTION STEP STEP RCTIVE
9RF=
FOR
BRfiE6 ~
N M R 3 NAHE T h P ~ 3 ST~
ai 1 Uelt - 8 Yea YesYeaYesYeaYes Yes Yes a5 . Reverse Flusn5 Yes YesYeaYesYeaYes Yes Yea a6 91 SIS To uaate 3 Yea YeaYesYesYesYeaYea Yes a7 13 113 To Colunn10 Yes YeaYeaYeaYesYesYea Yes a8 i0 iI8 To ~este 3 Yea YesYesYeaYesYeaYea Yes 49 a fait I9 Yaa YesYeaYeaYeaYesYea Yea 90 2 Reverse Fiuah5 Yea YesYeaYesYeaYesYea Yes ~
51 9 i18 To Column9 Yea YeaYeaYeaYesYeaYea Yes 52 3a Flush to IJaateS Yea YesYeaYeaYeaYeaYea Yes 53 9 I18 To Column9 Yea YeaYeaYeaYaaYesYea Yes 5i Z Reverse Flush5 Yes YeaYesYesYesYea~YSa Yea 55 3 t18 To Column9 Yes YesYeaYeaYeaYeaYea Yea 50 2 Reverse FlushS Yea YeaYeaYesYesYeaYes Yes 57 1 BLock FLusn 3 Yes YeaYeaYeaYeaYeaYes Yea 58 33 Cyele Entry I Yea YeaYeaYaaYeaYesYen Yes 93 3 318 To Column9 Yea YesYesYeaYeaYeaYea Yea 60 '_ Revnrae Flush5 Yea YesYeaYeaYesYesYea Yea 61 6 ~este-Port t Yea YeaYeaYeaYeaYssYea Yea 62 30 i17 To ~eate 3 Yes YesYeaYeaYeaYeaYea Yea 63 11 ill To Column7 Yea YeaYeaYeaYesYeaYea No 6a 3a Flush to 4leste1 Yea YeaYeaYeaYesYesYea No 65 II i17 To Column7 Yes YesYeaYesYaaYeaYea No 66 3a Flush to 4esteI Yaa YeaYesYeaYesYesYea No 67 II t17 To Column7 Yes YeaYeaYeaYaaYeaYaa No 68 34 Flush to ~eate1 Yes YeaYeaYeaYesYaaYea No 69 II i17 To Column7 Yes YesYesYeaYeaYesYea No 70 3a FLuah to IJesteI Yea YesYeaYesYeaYesYes No 71 11 i17 To Column7 Yas YeaYeaYesYeaYeaYea No 71 3a Flush to IJeate1 Yea YeaYeaYesYesYeaYaa No 73 11 ti7 To Column7 Yea YesYeaYaaYeaYeaYes Na 7a 3a Flush to ~eateS Yes YeaYesYeaYeaYaaYea No i 9 i18 To Column9 Yea YesYeeYesYeaYeaYes No = ~ Flush to 4laate7 Yes YeaYeaYea Yea No 76 3a Yea Yea 77 7 fiesta-Bottle1 Yas YesYeaYes Yea Yea Yea Yea 7 9 ti8 To Column9 Yea Yes Yaa Yes Yea Yes Yea Yea 8 2 Reverse FlushS Yea Yes Yea Yes 79 Yes Yea~Yea Yea 9 t18 To Column9 Yea Yea Yes Yea Yea Yea Yea Yea 80 2 Reverse FLuahS Yas Yes Yaa Yea HI Yes Yea Yea Yes 82 I Bloek Flush 3 Yea Yea Yea Yns Yea Yes Yea Yes 5$
NUMBER a NH11E -TIRE 9 ~ ~ T 5 5 -' T«
I 10 t18 To 4lsate 2 Yea YeaYeaYeaYeavesYes Yea 9 SIA To Column IS Yes YesYesYeaYeaYesYes Yes 3 ? Reverse Flush 20 Yea YesYeaYeaYesYesYes Yea a I Bloek FLush 6 Yes YeaYeaYeaYeaYesYea Yes I6 Cao Preo 10 Yea YeaYeaYeaYesYeaYea Yes a - . 30 Yea YeaYeaYeaYaaYeaYes Yes 91 Ceo To Column 7 10 a18 To ~aate 3 Yea YesYeaYeaYeaYesYea Yea B t Block Flush ~ Yes YeaYeaYaaYeaYeaYea Yes 9 4 Bait 300 Yea YesYeaYeaYeaYes.YeaYes I6 Cao Preo i0 Yea YeaYesYeaYeaYeaYea Yea 11 91 Cao To Column 30 Yes YesYeaYeaYeaYeaYea Yes I~ 10 i16 To Ueste~ 3 Yes YeaYeaYeaYeaYesYes Yes 13 I Bloek Fluan a Yea YesYeaYeaYeaYeaYea Yea la a Ueit 300 Yea YeaYesYeaYesYeaYes Yes 2 Reverse Fluan 10 Yea YeaYesYeaYeaYeaYea Yes 16 10 tIB To haste. 3 Yea YaaYnaYnaYeaYeaYea Yes 17 9 :18 To Column 15 Yea YaaYeaYeaYesYeaYes Yaa 18 2 Reverse Flush IA Yes YeaYesYesYaaYesYea Yes i9 9 t18 To Column IS Yna YeaYesYeaYeaYeaYea Yes 2 Reverse Flush t0 Yes YeaYesYeaYeaYnaYea Yea 21 9 it8 To Column 15 Yes YaaYnsYaaYeaYeaYes Yes 22 2 Reverse Flush I0 Yes YeaYeaYsaYeaYeaYaa Yea 23 9 t18 To Column 15 Yea YesYesYeaYesYeaYes Yas 26 2 Reverse Flush IB Yea YeaYeaYesYesYeaYea Yea 9 t18 To Column 15 Yea YeaYeaYeaYeaYeaYea Yes 25 2 Reverse Flush 6B Yes YeaYeaYeaYeaYesYes Yes Z7 I Bloek Fluan 5 Yea YesYeaYeaYesYeaYea Yea .... W093/13120 PCT/US92/11165 212492$
STEP FUNCTION STEP STEP RCTIVE SRFc FOR
BRSES
l9ER E NR11E TZhE R 6 C T c c ~ STc?
N
, S(j I 2 Reverse Flush60 Yes YeaYesYeaYeaYeaYea Yes Z Z7 i10 To CollectI7 Yea YeaYeaYaaYeaYeaYea Yes 3 10 SIB To 4laateS Yes YesYeaYeaYesYesYea Yes I Block Flush S Yea YeaYeaYaaYesYeaYea Yes S 6 4lelt 660 Yas YeaYeaYeaYeaYeaYea Yea 0 27 ti0 To Collect, Yea YesYeaYeaYeaYeaYes Yea ~~18 7 I0 SIB To ~aate S Yea YeaYesYesYeaYesYea Yea 8 1 Block Fluan S Yes YasYeaYaaYeaYeaYea Yea 9 ~ ~at,t 660 Yea YesYesYaaYesYeaYaa Yea 27 il0 To Collect18 Yes YesYeaYnaYeaYeaYes Yea II IB SIB To Wate S Yes YeaYeaYeaYeaYea'Yea Yea 1. I Block Fluan ~ Yea YeaYeaYesYesYesYes Yea IS t gait 660 Yea YesYeaYesYeaYesYea Yea la 27 i10 To Collect17 Yes YeaYesYeaYesYesYea Yea IS 10 i18 To ~aate S Yea YeaYeaYeaYeaYesYes Yea I6 t Block Flush S Yea YasYeaVaaYesYesYea Yea 17 s uut 660 Yes YeaYesYeaYaaYeaYea Yes IB 0 Flush To CLOT9 Yea YaaYeaYeaYeaYeaYaa Yes 19 Z7 i10 To Collectt4 Yea YeaYeaYeaYeaYeaYea Yea B Fluan To CLOT9 Yea YaaYeaYeaYeaYesYea Yea ZI . Raverae Flush60 Yes YeaYeaYesVaaYeaYes Yes 2Z 1 Block Flush Yaa YeaYaaYeaYeaYeaYea Yea Z5 10 i18 To Waste S Yaa YeaYaaYeaYaaYeaYea Yea Zi 9 SIB To Column30 Yea YeaYeaYeaYaaYeaYea Yes ZS 2 Reverse Flush60 Yna YasYeaYnaYaaYesYea Yea ZS I Fluan 18 Yea YesYesYeaYeaYesYes Yea 8loek . 2 Yea YeaYeaYeaYeaYeaYea Yea Z7 6Z s10 Vent WO 93/13120 PCT/US92/1 t 165 ~~~~~2g STEP FUNCTION STEP STEP RCTIV SRFE
FOR
i NR11E ThE R [ r 6 7 STEP
5-.
I '_8 Phoa Prec 10 Yes YeaYesYesYeaYesYea Yes 52 A To IJeate S Yes YesYeaYes~YesYeaYea Yes 3 .3 o To IJeate S Yea YeaYeaYeaYeaYeaYea Yes 6 56 C To 4leate 5 Yea YeaYeaYesYeaYeaYea Yes 5 E5 T To 4leste S Yea YeaYeaYeaYesYesYes Yea 6 ~ SS To Ylaate S Yea YeaYeaYeaYesYesYea Yea 7 57 36 To W ste S- Yea YeaYeaYesYesYeaYes Yes 8 58 Z7 To ~aate 6 Yea YesYesYeaYeaYeaYea Yes 9 61 TET To ~aate B Yea YeaYeaYesYeaYesYes Yes 10 10 i18 To ~este I0 Yes YesYeaYeaYesYeaYea Yes 11 Ifi Ceo Prep 10 Yes YesYesYeaYeaYeaYea Yes 1'_ .3 Cao A To ~aate5 Yea YeaYeaYesYesYeaYea Yea I~ 60 Cao 8 To hasteS Yes YeaYesYeaYes'feaYea Yes ii A1 SIS To ~eate 8 Yea YeaYeaYeaYesYeaYes Yes 15 A2 it1 To ~este 8 Yes YeaYeaYeaYeaYesYea Yes I6 30 i17 To ~aate 10 Yes YeaYeaYea.YeaYesYea- Yes 17 18 i18 To Weate 15 Yea YsaYeaYeaYesYesYea Yea 18 1 8loek Fluah IS Yea YeaYeaYeaYesYesYea Yes WO 93/13120 ~ ~ 2 4 9 2 8 PCT/US92/11165 6'- 6bT 6TT Tb6 TT6 TT6 TF6 TT6 TT6 TT6 TT6 TT6 TT6 TT6 TT6 TT6 TT6 TT6 TTT TTT TTT TTT TTT .TTT TT -3' VERSION Z.00 SEQUENCE NRIIE:t S%-2 SEQUENCE LEH6TM:10 OATE: Au0 27.
TI!: ta:06 COHHENT:
S'- ?7T bAC T65 T -3
Claims (10)
1. A synthetic oligonucleotide useful as an amplifier probe in a sandwich hybridization assay for Hepatitis B Virus (HBV) comprising a first segment comprising a nucleotide sequence substantially complementary to a segment of HBV
nucleic acid; and a second segment comprising a nucleotide sequence substantially complementary to an oligonucleotide multimer and substantially non-complementary to said segment of HBV nucleic acid;
wherein said nucleotide sequence substantially complementary to a segment of HBV nucleic acid is selected from the group consisting of:
TTGTGGGTCTTTTGGGYTTTGCTGCYCCWT(SEQ ID N0:6), CCTKCTCGTGTTACAGGCGGGGTTTTTCTT(SEQ ID N0:7), TCCATGGCTGCTAGGSTGTRCTGCCAACTG(SEQ ID NO:8), GCYTAYAGACCACCAAATGCCCCTATCYTA(SEQ ID N0:9), CTGTTCAAGCCTCCAAGCTGTGCCTTGGGT (SEQ ID NO:10), CATGGAGARCAYMACATCAGGATTCCTAGG (SEQ ID N0:11), TCCTGGYTATCGCTGGATGTGTCTGCGGCGT (SEQ ID N0:12), GGCGCTGAATCCYGCGGACGACCCBTCTCG (SEQ ID N0:13), CTTCGCTTCACCTCTGCACGTHGCATGGMG (SEQ ID N0:14), GGTCTSTGCCAAGTGTTTGCTGACGCAACC (SEQ ID NO:15), CCTKCGCGGGACGTCCTTTGTYTACGTCCC (SEQ ID N0:16), MCCTCTGCCTAATCATCTCWTGTWCATGTC (SEQ ID N0:17), CGACCACGGGGCGCACCTCTCTTTACGCGG (SEQ ID N0:18), TGCCCAAGGTCTTACAYAAGAGGACTCTTG (SEQ ID N0:19), CGTCAATCTYCKCGAGGACTGGGGACCCTG (SEQ ID N0:20), ATGTTGCCCGTTTGTCCTCTAMTTCCAGGA (SEQ ID N0:21), ATCTTCTTRTTGGTTCTTCTGGAYTAYCAA (SEQ ID N0:22), ATCATMTTCCTCTTCATCCTGCTGCTATGC (SEQ ID N0:23), CAATCACTCACCAACCTCYTGTCCTCCAAY (SEQ ID N0:24), GTGTCYTGGCCAAAATTCGCAGTCCCCAAC (SEQ ID N0:25), CTCGTGGTGGACTTCTCTCAATTTTTCTAGG (SEQ ID N0:26) , GACAAGAATCCTCACAATACCRCAGAGTCT (SEQ ID N0:27), TTTTGGGGTGGAGCCCKCAGGCTCAGGGCR (SEQ ID N0:28), CACCATATTCTTGGGAACAAGAKCTACAGC (SEQ ID N0:29), ACACTTCCGGARACTACTGTTGTTAGACGA (SEQ ID NQ:30), GTVTCTTTYGGAGTGTGGATTCGCACTCCT (SEQ ID N0:31), TTGGAGCWWTCGTGGAGTTACTCTCKTTTT (SEQ ID N0:32), TTTGGGGCATGGACATYGAYCCKTATAAAG (SEQ ID N0:33), AAWGRTCTTTGTAYTAGGAGGCTGTAGGCA (SEQ ID N0:34), RGACTGGGAGGAGYTGGGGGAGGAGATTAG (SEQ ID N0:35), CCTTGAGGCMTACTTCAAAGACTGTKTGTT (SEQ ID N0:36), GTCTGTGCCTTCTCATCTGCCGGWCCGTGT (SEQ ID N0:37), AGCMGCTTGTTTTGCTCGCAGSMGGTCTGG (SEQ ID N0:38), GGCTCSTCTGCCGATCCATACTGCGGAACT (SEQ ID N0:39), MTKAACCTTTACCCCGTTGCTCGGCAACGG (SEQ ID N0:40), GTGGCTCCAGTTCMGGAACAGTAAACCCTG (SEQ ID N0:41), , KAARCAGGCTTTYACTTTCTCGCCAACTTA (SEQ ID N0:42), CCTCCKCCTGCCTCYACCAATCGSCAGTCA (SEQ ID N0:43), ACCAATTTTCTTYTGTCTYTGGGTATACAT (SEQ ID N0:44).
nucleic acid; and a second segment comprising a nucleotide sequence substantially complementary to an oligonucleotide multimer and substantially non-complementary to said segment of HBV nucleic acid;
wherein said nucleotide sequence substantially complementary to a segment of HBV nucleic acid is selected from the group consisting of:
TTGTGGGTCTTTTGGGYTTTGCTGCYCCWT(SEQ ID N0:6), CCTKCTCGTGTTACAGGCGGGGTTTTTCTT(SEQ ID N0:7), TCCATGGCTGCTAGGSTGTRCTGCCAACTG(SEQ ID NO:8), GCYTAYAGACCACCAAATGCCCCTATCYTA(SEQ ID N0:9), CTGTTCAAGCCTCCAAGCTGTGCCTTGGGT (SEQ ID NO:10), CATGGAGARCAYMACATCAGGATTCCTAGG (SEQ ID N0:11), TCCTGGYTATCGCTGGATGTGTCTGCGGCGT (SEQ ID N0:12), GGCGCTGAATCCYGCGGACGACCCBTCTCG (SEQ ID N0:13), CTTCGCTTCACCTCTGCACGTHGCATGGMG (SEQ ID N0:14), GGTCTSTGCCAAGTGTTTGCTGACGCAACC (SEQ ID NO:15), CCTKCGCGGGACGTCCTTTGTYTACGTCCC (SEQ ID N0:16), MCCTCTGCCTAATCATCTCWTGTWCATGTC (SEQ ID N0:17), CGACCACGGGGCGCACCTCTCTTTACGCGG (SEQ ID N0:18), TGCCCAAGGTCTTACAYAAGAGGACTCTTG (SEQ ID N0:19), CGTCAATCTYCKCGAGGACTGGGGACCCTG (SEQ ID N0:20), ATGTTGCCCGTTTGTCCTCTAMTTCCAGGA (SEQ ID N0:21), ATCTTCTTRTTGGTTCTTCTGGAYTAYCAA (SEQ ID N0:22), ATCATMTTCCTCTTCATCCTGCTGCTATGC (SEQ ID N0:23), CAATCACTCACCAACCTCYTGTCCTCCAAY (SEQ ID N0:24), GTGTCYTGGCCAAAATTCGCAGTCCCCAAC (SEQ ID N0:25), CTCGTGGTGGACTTCTCTCAATTTTTCTAGG (SEQ ID N0:26) , GACAAGAATCCTCACAATACCRCAGAGTCT (SEQ ID N0:27), TTTTGGGGTGGAGCCCKCAGGCTCAGGGCR (SEQ ID N0:28), CACCATATTCTTGGGAACAAGAKCTACAGC (SEQ ID N0:29), ACACTTCCGGARACTACTGTTGTTAGACGA (SEQ ID NQ:30), GTVTCTTTYGGAGTGTGGATTCGCACTCCT (SEQ ID N0:31), TTGGAGCWWTCGTGGAGTTACTCTCKTTTT (SEQ ID N0:32), TTTGGGGCATGGACATYGAYCCKTATAAAG (SEQ ID N0:33), AAWGRTCTTTGTAYTAGGAGGCTGTAGGCA (SEQ ID N0:34), RGACTGGGAGGAGYTGGGGGAGGAGATTAG (SEQ ID N0:35), CCTTGAGGCMTACTTCAAAGACTGTKTGTT (SEQ ID N0:36), GTCTGTGCCTTCTCATCTGCCGGWCCGTGT (SEQ ID N0:37), AGCMGCTTGTTTTGCTCGCAGSMGGTCTGG (SEQ ID N0:38), GGCTCSTCTGCCGATCCATACTGCGGAACT (SEQ ID N0:39), MTKAACCTTTACCCCGTTGCTCGGCAACGG (SEQ ID N0:40), GTGGCTCCAGTTCMGGAACAGTAAACCCTG (SEQ ID N0:41), , KAARCAGGCTTTYACTTTCTCGCCAACTTA (SEQ ID N0:42), CCTCCKCCTGCCTCYACCAATCGSCAGTCA (SEQ ID N0:43), ACCAATTTTCTTYTGTCTYTGGGTATACAT (SEQ ID N0:44).
2. The synthetic oligoaucleotide of claim 1, wherein said second segment comprises AGGCATAGGACCCGTGTCTT (SEQ ID N0:54).
3. A synthetic oligonucleotide useful as a capture probe in a sandwich hybridization assay for HBV
comprising a first segment comprising a nucleotide sequence substantially complementary to a segment of HBV
nucleic acid; and a second segment comprising a nucleotide sequence substantially complementary to an oligonucleotide bound to a solid phase and substantially non-complementary to said segment of HBV nucleic acid, wherein said nucleotide sequence substantially complementary to a segment of HBV nucleic acid is selected from the group consisting of:
TATTCCCATCCCATCRTCCTGGGCTTTCGS (SEQ ID NO:45), TATATGGATGATGTGGTATTGGGGGCCAAG (SEQ ID NO:46), CGTAGGGCTTTCCCCCACTGTTTGGCTTTC (SEQ ID NO:47), GCTCAGTTTACTAGTGCCATTTGTTCAGTG (SEQ ID NO;48), CCTATGGGAGRGGGCCTG,GYCCGTTTCTC (SEQ ID NO:49), GTCCCCTAGAAGAAGAACTCCCTCGCCTCG (SEQ ID NO:50), ACGMAGRTCTCMATCGCCGCGTCGCAGAAGA (SEQ ID NO:51), CAATCTCGGGAATCTCAATGTTAGTATYCC (SEQ ID NO:52), GACTCATAAGGTSGGRAACTTTACRGGGCT (SEQ ID N:53).
comprising a first segment comprising a nucleotide sequence substantially complementary to a segment of HBV
nucleic acid; and a second segment comprising a nucleotide sequence substantially complementary to an oligonucleotide bound to a solid phase and substantially non-complementary to said segment of HBV nucleic acid, wherein said nucleotide sequence substantially complementary to a segment of HBV nucleic acid is selected from the group consisting of:
TATTCCCATCCCATCRTCCTGGGCTTTCGS (SEQ ID NO:45), TATATGGATGATGTGGTATTGGGGGCCAAG (SEQ ID NO:46), CGTAGGGCTTTCCCCCACTGTTTGGCTTTC (SEQ ID NO:47), GCTCAGTTTACTAGTGCCATTTGTTCAGTG (SEQ ID NO;48), CCTATGGGAGRGGGCCTG,GYCCGTTTCTC (SEQ ID NO:49), GTCCCCTAGAAGAAGAACTCCCTCGCCTCG (SEQ ID NO:50), ACGMAGRTCTCMATCGCCGCGTCGCAGAAGA (SEQ ID NO:51), CAATCTCGGGAATCTCAATGTTAGTATYCC (SEQ ID NO:52), GACTCATAAGGTSGGRAACTTTACRGGGCT (SEQ ID N:53).
4. The synthetic oligonucleotide of claim 3, wherein said second segment is CTTCTTTGGAGAAAGTGGTG (SEQ ID NO:55).
5. A set of synthetic oligonucleotides useful as amplifier probes in a sandwich hybridization assay for HBV, comprising two oligonucleotides, wherein each member of the set comprises a first segment comprising a nucleotide sequence substantially complementary to a segment of HBV
nucleic acid; and a second segment comprising a nucleotide sequence substantially complementary to an oligonucleotide multimer and substantially non-complementary to said segment of HBV nucleic acid, wherein said nucleotide sequence substantially complementary to a segment of HBV nucleic acid is selected from the group consisting of:
TTGTGGGTCTTTTGGGYTTTGCTGCYCCWT (SEQ ID NO:6), CCTKCTCGTGTTACAGGCGGGGTTTTTCTT (SEQ ID NO:7), TCCATGGCTGCTAGGSTGTRCTGCCAACTG (SEQ ID NO:8), GCYTAYAGACCACCAAATGCCCCTATCYTA (SEQ ID NO:9), CTGTTCAAGCCTCCAAGCTGTGCCTTGGGT (SEQ ID NO:10), CATGGAGARCAYMACATCAGGATTCCTAGG (SEQ ID NO:11), TCCTGGYTATCGCTGGATGTGTCTGCGGCGT (SEQ ID NO:12), GGCGCTGAATCCYGCGGACGACCCBTCTCG (SEQ ID NO:13), CTTCGCTTCACCTCTGCACGTAGCATGGMG (SEQ ID NO:14), GGTCTSTGCCAAGTGTTTGCTGACGCAACC (SEQ ID NO:15), CCTKCGCGGGACGTCCTTTGTYTACGTCCC (SEQ ID NO:16), MCCTCTGCCTAATCATCTCWTGTWCATGTC (SEQ ID NO:17), CGACCACGGGGCGCACCTCTCTTTACGCGG (SEQ ID NO:18), TGCCCAAGGTCTTACAYAAGAGGACTCTTG (SEQ ID NO:19), CGTCAATCTYCKCGAGGACTGGGGACCCTG (SEQ ID NO:20), ATGTTGCCCGTTTGTCCTCTAMTTCCAGGA (SEQ ID NO:21), ATCTTCTTRTTGGTTCTTCTGGAYTAYCAA (SEQ ID NO:22), ATCATMTTCCTCTTCATCCTGCTGCTATGC (SEQ ID NO:23), CAATCACTCACCAACCTCYTGTCCTCCAAY (SEQ ID NO:24), GTGTCYTGGCCAAAATTCGCAGTCCCCAAC (SEQ ID NO:25), CTCGTGGTGGACTTCTCTCTIATTTCTAGG (SEQ ID NO:26), GACAAGAATCCTCACAATACCRCAGAGTCT (SEQ ID NO:27), TTTTGGGGTGGAGCCCRCAGGCTCAGGGCR (SEQ ID NO:28), CACCATATTCTTGGGAACAAGARCTACAGC (SEQ ID NO:29), ACACTTCCGGARACTACTGTTGTTAGACGA (SEQ ID NO:3O), GTVTCTITYGGAGTGTGGATTCGCACTCCT (SEQ ID NO:31), TTGGAGCWWCTGTGGAGTTACTCTCRTTTT (SEQ ID NO:32), TTTGGGGCATGGACATYGAYCCRTATAAAG (SEQ ID NO:33), AAWGRTCTTTGTAYTAGGAGGC1GTAGGCA (SEQ ID NO:34), RGACTGGGAGGAGYTGGGGGAGGAGATTAG (SEQ ID NO:35), CCTTGAGGCMTACTTCAAAGACTGTR1GTT (SEQ ID NO:36), GTCTGTGCCTTCTCATCTGCCGGWCCGTGT (SEQ ID NO:37), AGCMGCTTGTTTTGCTCGCAGSMGGTC1GG (SEQ ID NO:38), GGCTCSTCTGCCGATCCATAC1GCGGAACT (SHQ ID NO:39), MTKAACCTTTACCCCGTTGCTCGGCAACGG (SEQ ID NO:4O), GTGGCTCCAGTTCMGGAACAGTAAACCCTG (SEQ ID NO:41), RAARCAGGCTTTYACTTTCTCGCCAACITA (SEQ ID NO:42), CCTCCRCCTGCCTCYACCAATCGSCAGTCA (SEQ ID NO:43), ACCAATTTTCTTYTGTCTYTGGGTATACAT (SHQ ID NO:44).
nucleic acid; and a second segment comprising a nucleotide sequence substantially complementary to an oligonucleotide multimer and substantially non-complementary to said segment of HBV nucleic acid, wherein said nucleotide sequence substantially complementary to a segment of HBV nucleic acid is selected from the group consisting of:
TTGTGGGTCTTTTGGGYTTTGCTGCYCCWT (SEQ ID NO:6), CCTKCTCGTGTTACAGGCGGGGTTTTTCTT (SEQ ID NO:7), TCCATGGCTGCTAGGSTGTRCTGCCAACTG (SEQ ID NO:8), GCYTAYAGACCACCAAATGCCCCTATCYTA (SEQ ID NO:9), CTGTTCAAGCCTCCAAGCTGTGCCTTGGGT (SEQ ID NO:10), CATGGAGARCAYMACATCAGGATTCCTAGG (SEQ ID NO:11), TCCTGGYTATCGCTGGATGTGTCTGCGGCGT (SEQ ID NO:12), GGCGCTGAATCCYGCGGACGACCCBTCTCG (SEQ ID NO:13), CTTCGCTTCACCTCTGCACGTAGCATGGMG (SEQ ID NO:14), GGTCTSTGCCAAGTGTTTGCTGACGCAACC (SEQ ID NO:15), CCTKCGCGGGACGTCCTTTGTYTACGTCCC (SEQ ID NO:16), MCCTCTGCCTAATCATCTCWTGTWCATGTC (SEQ ID NO:17), CGACCACGGGGCGCACCTCTCTTTACGCGG (SEQ ID NO:18), TGCCCAAGGTCTTACAYAAGAGGACTCTTG (SEQ ID NO:19), CGTCAATCTYCKCGAGGACTGGGGACCCTG (SEQ ID NO:20), ATGTTGCCCGTTTGTCCTCTAMTTCCAGGA (SEQ ID NO:21), ATCTTCTTRTTGGTTCTTCTGGAYTAYCAA (SEQ ID NO:22), ATCATMTTCCTCTTCATCCTGCTGCTATGC (SEQ ID NO:23), CAATCACTCACCAACCTCYTGTCCTCCAAY (SEQ ID NO:24), GTGTCYTGGCCAAAATTCGCAGTCCCCAAC (SEQ ID NO:25), CTCGTGGTGGACTTCTCTCTIATTTCTAGG (SEQ ID NO:26), GACAAGAATCCTCACAATACCRCAGAGTCT (SEQ ID NO:27), TTTTGGGGTGGAGCCCRCAGGCTCAGGGCR (SEQ ID NO:28), CACCATATTCTTGGGAACAAGARCTACAGC (SEQ ID NO:29), ACACTTCCGGARACTACTGTTGTTAGACGA (SEQ ID NO:3O), GTVTCTITYGGAGTGTGGATTCGCACTCCT (SEQ ID NO:31), TTGGAGCWWCTGTGGAGTTACTCTCRTTTT (SEQ ID NO:32), TTTGGGGCATGGACATYGAYCCRTATAAAG (SEQ ID NO:33), AAWGRTCTTTGTAYTAGGAGGC1GTAGGCA (SEQ ID NO:34), RGACTGGGAGGAGYTGGGGGAGGAGATTAG (SEQ ID NO:35), CCTTGAGGCMTACTTCAAAGACTGTR1GTT (SEQ ID NO:36), GTCTGTGCCTTCTCATCTGCCGGWCCGTGT (SEQ ID NO:37), AGCMGCTTGTTTTGCTCGCAGSMGGTC1GG (SEQ ID NO:38), GGCTCSTCTGCCGATCCATAC1GCGGAACT (SHQ ID NO:39), MTKAACCTTTACCCCGTTGCTCGGCAACGG (SEQ ID NO:4O), GTGGCTCCAGTTCMGGAACAGTAAACCCTG (SEQ ID NO:41), RAARCAGGCTTTYACTTTCTCGCCAACITA (SEQ ID NO:42), CCTCCRCCTGCCTCYACCAATCGSCAGTCA (SEQ ID NO:43), ACCAATTTTCTTYTGTCTYTGGGTATACAT (SHQ ID NO:44).
6. The set of synthetic oligonucleotides of claim 5, wherein said second segment comprises AGGCATAGGACCCGTGTCTT (SEQ ID NO:54).
7. A set of synthetic oligonucleotides useful as capture probes in a sandwich hybridization assay for HBV, comprising two oligonucleotides, wherein each member of the set comprises a first segment comprising a nucleotide sequence substantially complementary to a segment of HBV nucleic acid; and a second segment comprising a nucleotide sequence substantially complementary to an oligonucleotide bound to a solid phase and substantially non-complementary to said segment of HBV
nucleic acid, wherein said nucleotide sequence substantially complementary to a segment of HBV nucleic acid is selected from the group consisting of:
TATTCCCATCCCATCRTCCTGGGCTTTCGS (SEQ ID NO;45), TATATGGATGATGTGGTATTGGGGGCCAAG (SEQ ID NO:46), CGTAGGGCTTTCCCCCACTGTTTGGCTTTC (SEQ ID NO:47), GCTCAGTTTACTTTGTGCCATTGTTCAGTG (SEQ ID NO:48), CCTATGGGAGXGGGGGTCAGYCCGTITCTC (SEQ ID NO:49), ACGMAGRTCTCMAGTCCCCTGGGTCACTCG (SEQ ID NO:50), ACGMAGRTCTCATCGCCCGCGCCGCAGAAGA (SEQ ID NO:51), CAATCTCGGGAATCTCAATGTTATGATYCC (SEQ ID NO:52), GACTCATAATGGTSGGRAACTTTAKGGGCT (SEQ ID NO:53).
nucleic acid, wherein said nucleotide sequence substantially complementary to a segment of HBV nucleic acid is selected from the group consisting of:
TATTCCCATCCCATCRTCCTGGGCTTTCGS (SEQ ID NO;45), TATATGGATGATGTGGTATTGGGGGCCAAG (SEQ ID NO:46), CGTAGGGCTTTCCCCCACTGTTTGGCTTTC (SEQ ID NO:47), GCTCAGTTTACTTTGTGCCATTGTTCAGTG (SEQ ID NO:48), CCTATGGGAGXGGGGGTCAGYCCGTITCTC (SEQ ID NO:49), ACGMAGRTCTCMAGTCCCCTGGGTCACTCG (SEQ ID NO:50), ACGMAGRTCTCATCGCCCGCGCCGCAGAAGA (SEQ ID NO:51), CAATCTCGGGAATCTCAATGTTATGATYCC (SEQ ID NO:52), GACTCATAATGGTSGGRAACTTTAKGGGCT (SEQ ID NO:53).
8. The set of synthetic oligonucleotides of claim 7, wherein said second segment comprises CTTCTTTGGAGAAAGTGGTG (SEQ ID NO:55) .
9. A solution sandwich hybridization assay for detecting the presence of HBV in a sample, comprising (a) contacting the sample under conditions of specific hybridization with (i) an excess of amplifier probes comprising the set of synthetic oligonucleotides of claim 5 and (ii) an excess of capture probes comprising the set of synthetic oligonucleotides of claim 7;
(b) contacting the product of step (a) under conditions of specific hybridization with said oligonucleotide bound to the solid phase;
(c) thereafter separating materials not bound to the solid phase;
(d) contacting the bound product of step (c) under conditions of specific hybridization with a nucleic acid multimer, said multimer comprising at least one oligonucleotide unit that is substantially complementary to the second segment of the amplifier probe polynucleotide and a multiplicity of second oligonucleotide units that are substantially complementary to a labeled oligonucleotide;
(e) removing unbound multimer;
(f) contacting under conditions of specific hybridization the solid phase complex product of step (e) with the labeled oligonucleotide;
(g) removing unbound labeled oligonucleotide; and (h) detecting the presence of label in the solid phase complex product of step (g).
(b) contacting the product of step (a) under conditions of specific hybridization with said oligonucleotide bound to the solid phase;
(c) thereafter separating materials not bound to the solid phase;
(d) contacting the bound product of step (c) under conditions of specific hybridization with a nucleic acid multimer, said multimer comprising at least one oligonucleotide unit that is substantially complementary to the second segment of the amplifier probe polynucleotide and a multiplicity of second oligonucleotide units that are substantially complementary to a labeled oligonucleotide;
(e) removing unbound multimer;
(f) contacting under conditions of specific hybridization the solid phase complex product of step (e) with the labeled oligonucleotide;
(g) removing unbound labeled oligonucleotide; and (h) detecting the presence of label in the solid phase complex product of step (g).
10. A kit for the detection of HBV in a sample comprising in combination (i) amplifier probes comprising the set of synthetic oligonucleotides of claim 5;
(ii) capture probes comprising the set of synthetic oligonucleotides of claim 7;
(iii) a nucleic acid multimer, said multimer comprising at least one oligonucleotide unit that is substantially complementary to the second segment of the amplifier probe polynucleotide and a multiplicity of second oligonucleotide units that are substantially complementary to a labeled oligonucleotide; and Iv) a labeled oligonucleotide.
(ii) capture probes comprising the set of synthetic oligonucleotides of claim 7;
(iii) a nucleic acid multimer, said multimer comprising at least one oligonucleotide unit that is substantially complementary to the second segment of the amplifier probe polynucleotide and a multiplicity of second oligonucleotide units that are substantially complementary to a labeled oligonucleotide; and Iv) a labeled oligonucleotide.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US813,586 | 1977-07-07 | ||
| US81358691A | 1991-12-23 | 1991-12-23 | |
| PCT/US1992/011165 WO1993013120A1 (en) | 1991-12-23 | 1992-12-22 | Hbv amplifier probes for use in solution phase sandwich hybridization assays |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2124928A1 CA2124928A1 (en) | 1993-07-08 |
| CA2124928C true CA2124928C (en) | 2003-06-17 |
Family
ID=25212830
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002124928A Expired - Fee Related CA2124928C (en) | 1991-12-23 | 1992-12-22 | Hbv amplifier probes for use in solution phase sandwich hybridization assays |
Country Status (8)
| Country | Link |
|---|---|
| US (1) | US5736316A (en) |
| EP (1) | EP0618924B1 (en) |
| JP (1) | JPH07502653A (en) |
| AT (1) | ATE199158T1 (en) |
| CA (1) | CA2124928C (en) |
| DE (1) | DE69231693T2 (en) |
| TW (1) | TW293845B (en) |
| WO (1) | WO1993013120A1 (en) |
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| US5985662A (en) * | 1995-07-13 | 1999-11-16 | Isis Pharmaceuticals Inc. | Antisense inhibition of hepatitis B virus replication |
| ZA973367B (en) * | 1996-04-19 | 1997-11-18 | Innogenetics Nv | Method for typing and detecting HBV. |
| ES2246546T3 (en) * | 1997-12-12 | 2006-02-16 | Digene Corporation | ASSESSMENT OF DISEASES RELATED TO THE HUMAN PAPILOMA VIRUS. |
| US6203989B1 (en) | 1998-09-30 | 2001-03-20 | Affymetrix, Inc. | Methods and compositions for amplifying detectable signals in specific binding assays |
| US6759193B2 (en) * | 1999-07-28 | 2004-07-06 | The Government Of The Republic Of Singapore | Detection of human hepatitis B virus surface antigen mutants by specific amplification and its application on gene chip |
| US7439016B1 (en) | 2000-06-15 | 2008-10-21 | Digene Corporation | Detection of nucleic acids by type-specific hybrid capture method |
| US7601497B2 (en) | 2000-06-15 | 2009-10-13 | Qiagen Gaithersburg, Inc. | Detection of nucleic acids by target-specific hybrid capture method |
| SG90149A1 (en) * | 2000-07-18 | 2002-07-23 | Government Of The Republic Of | Diagnostic assay |
| US20020081589A1 (en) * | 2000-10-12 | 2002-06-27 | Jing-Shan Hu | Gene expression monitoring using universal arrays |
| US6583279B1 (en) | 2001-01-26 | 2003-06-24 | Becton, Dickinson And Company | Sequences and methods for detection of hepatitis B virus |
| JP2004525632A (en) * | 2001-03-07 | 2004-08-26 | バイオメリオ・ベー・ベー | Amplification and detection of HBV DNA using transcriptional amplification |
| US20030087256A1 (en) * | 2001-04-06 | 2003-05-08 | Micrologix Biotech Inc. | Thiophosphate nucleic acid-based compounds |
| WO2003031934A2 (en) * | 2001-10-09 | 2003-04-17 | Chiron Corporation | Identification of oligonucleotides for the capture, detection and quantitation of hepatitis b viral dna |
| US7015317B2 (en) * | 2002-05-02 | 2006-03-21 | Abbott Laboratories | Polynucleotides for the detection and quantification of hepatitis B virus nucleic acids |
| AU2003253651C1 (en) * | 2002-06-14 | 2010-06-03 | Gen-Probe Incorporated | Compositions and methods for detecting hepatitis B virus |
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| FR2444713A1 (en) * | 1978-12-18 | 1980-07-18 | Pasteur Institut | PROCESS FOR PRODUCING DNA COMPRISING THE GENOME OF HEPATITIS B VIRUS AND VECTOR COMPRISING SAME |
| US4562159A (en) * | 1981-03-31 | 1985-12-31 | Albert Einstein College Of Medicine, A Division Of Yeshiva Univ. | Diagnostic test for hepatitis B virus |
| EP0068719A3 (en) * | 1981-06-16 | 1984-03-28 | Takeda Chemical Industries, Ltd. | Deoxyribonucleic acids, their production and use |
| US5008182A (en) * | 1986-01-10 | 1991-04-16 | Cetus Corporation | Detection of AIDS associated virus by polymerase chain reaction |
| US4868105A (en) * | 1985-12-11 | 1989-09-19 | Chiron Corporation | Solution phase nucleic acid sandwich assay |
| EP0278940A3 (en) * | 1987-01-30 | 1988-12-07 | Smithkline Biologicals S.A. | Hepatitis b virus surface antigens and hybrid antigens containing them |
| US5124246A (en) * | 1987-10-15 | 1992-06-23 | Chiron Corporation | Nucleic acid multimers and amplified nucleic acid hybridization assays using same |
| CA1339351C (en) * | 1987-10-15 | 1997-08-26 | Michael S. Urdea | Nucleic acid multimers and amplified nucleic acid hybridization assays using same |
| ATE174339T1 (en) * | 1990-07-27 | 1998-12-15 | Chiron Corp | LARGE CAMB-SHAPED BRANCHED POLYNUCLEOTIDES |
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1992
- 1992-12-22 AT AT93902719T patent/ATE199158T1/en not_active IP Right Cessation
- 1992-12-22 CA CA002124928A patent/CA2124928C/en not_active Expired - Fee Related
- 1992-12-22 WO PCT/US1992/011165 patent/WO1993013120A1/en active IP Right Grant
- 1992-12-22 DE DE69231693T patent/DE69231693T2/en not_active Expired - Lifetime
- 1992-12-22 EP EP93902719A patent/EP0618924B1/en not_active Expired - Lifetime
- 1992-12-22 JP JP5511870A patent/JPH07502653A/en active Pending
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1993
- 1993-01-20 TW TW082100428A patent/TW293845B/zh not_active IP Right Cessation
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1994
- 1994-01-24 US US08/186,229 patent/US5736316A/en not_active Expired - Lifetime
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| EP0618924A1 (en) | 1994-10-12 |
| JPH07502653A (en) | 1995-03-23 |
| DE69231693T2 (en) | 2001-05-31 |
| ATE199158T1 (en) | 2001-02-15 |
| EP0618924B1 (en) | 2001-02-14 |
| CA2124928A1 (en) | 1993-07-08 |
| WO1993013120A1 (en) | 1993-07-08 |
| US5736316A (en) | 1998-04-07 |
| EP0618924A4 (en) | 1996-01-17 |
| TW293845B (en) | 1996-12-21 |
| DE69231693D1 (en) | 2001-03-22 |
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