CA2186465A1 - Hybridization-ligation assays for the detection of specific nucleic acid sequences - Google Patents

Abstract

A new method has been developed for conducting a gene probe assay. The preferred technique involves (1) using a gene amplification technique (e.g., PCR) to multiply the gene sequence of interest and (2) using a hybridization-ligation detection methodology, wherein the sequences of probes hybridized to the target sequence allow for separation and detection (e.g., probes might contain a combination of magnetic particles and acridinium esters) to determine if a specific sequence is present,

Description

W095/27078 2186465 r~

HYBRIDIZATION - LIGATIOM ASSAYS FOR THE
DETECTION OF SPECIFIC NUCLEIC ACID SEQUENCES

Background of the Invention Gene probe techniques have become an important analytical tool for predicting the inr;~nre of inherited disease and in diagnosing existing medical conditions.
However, the currently-used techniques are 510w, laborious and involve the use of harmful chemicals. Some procedures used in the gene probe f ield were discussed recently by Plaha et al (14 BioTechniques 566, 1993). Current gene probe techniriues typically involve the use of electrophoresis, frequently on long polyacrylamide gel6.
Several of the chemicals to which the lab personnel are exposed are rr~nq; rl~red to be hazardous . Namely, the acrylamide monomer, some of which may remain in the polymeric gel, is considered to be a neurotoxin. The ethidium bromide used as the staining agent is a mutagen.
Polyacrylamide gel electrophoresis typically requires several hours, while the analysis using the new gels described in Plaha require 16 hours for a single run.
Other techniques use radioactive markers, which re~uire the use of special h~n~ll; n~ controls and disposal techniques. Also, the other aspects of the current procedures reriuire a relatively long period of time. (For W0 95/27078 2 1 8 6 ~ 6 5 example, the Southern Blot procedure requires about 48 hours to complete. ) Furthermore, the electrophoresis technique itself may produce uncertain results. First, ethidium bromide tf-rhn;~r~ p are often not very sensitive. Second, the conditions to adequately separate the different gene LL _ P must be developed. Third, the results are nearly always qualitative, not quantitative. In addition, the inability to distinguish LL _ tR of DNA whic_ are the same or similar size but differ in sequence, even by a single base, also limits the usefulness of past procedures .
~imitations o~ the sensitivity inherent to DNA based assays may be UVt:l~ - by the polymerase chain reaction (PCR) . The amplification of a specific sequence by PCR
enables the detection of that seS~uence when present in a sample in extremely low amounts (Saiki, et al., 230 Science 1350, 1985). Although the PCR t~rhn;~l~ can amplify DNA se~uences to uv~L~' - the sensitivity limitations that existed before ~CR was available, a number of problems remain attendant with the use of PCR.
The products of a PCR reaction often include artifacts due to primer-dimers and- non-specific priming events, especially in the absence of the target sequence in the sample. By virtue of its exquisite sensitivity, PCR is susceptible to false positive results due to rrnt?lm;n~tion. The PCR technique itself does not readily allow the discrimination of small differences between sequences such as point mutations which may underly genetic disea8es, such as in cystic fibrosis. As a consequence of these limitations, verif ication of the presence of the specific target sequence af~er W0 95R7078 r ., amplification by PCR i8 a desirable if not an ~ nt;i~1 step in a DNA assay.
Earlier workers have used hybr;tl;7~t;r~n and ligation techniques as a precursor to analyzing samples on various gels. (See, Landegren et al, 241 Science 1077, 1988).
However, these techniques are slow, inconvenient and are not amenable to usage on automated instruments.
A novel analytical method has been developed, which l~l;m;n;~t.o~ the drawbackg of the current techniques for the analysis of DNA sequences and provides quicker and more accurate results. The instant novel technique can be l]t; l; ~Pd along with PCR to improve accuracy in gene probe assays.
Summary of Invention A new method has been developed for ~nrlll~t;n~ a gene probe assay . The pref erred technique involves ( 1 ) using a gene amplification technique (e.g., PCR) to multiply the gene sequence of interest and (2) using a hybridization -ligation detection I th~ )l ogy, wherein the sequences of probes hybridized to the target sequence allow for separation and detection (e.g., probes might contain a ' ;n::lt;~n of magnetic particles and acridinium esters) to determine if a specif ic sequence is present .

Brief Description of the Drawings Figure 1: The probe a6say formats used for the detection of the delta F-508 and normal alleles.

wo 95f27078 2 18 6 ~ 6 5 ~ c - - ~
Figure 2: The results of the HLM for the simultaneous rl~t.-~t;~n of delta F-508 and normal alleles in nine samples of human DNA that have been amplified by PCR. The probe specific for the delta F-508 allele was labeled with DMAE and the probe specific for the normal allele was lableled with LE:A13.
Figure 3: A portion of the sequence of exon 10 of the CFTR gene in the vicinity of the sltes for the delta F-508 and delta I-507 mutations. SeSIuences underlined are complementary to the sequences of probes immobilized on paramagnetic particles (PMP.508 or PMP.507)and labeled wi th acridinium e 8 t er ( 5 0 8 . NOR or~ 5 0 7 . NOR ) .
Figure 4: The hybrids formed between the probes for the delta F-508 assay and delta I-507 assay and the various alleles .
Figure 5: HLM results for the detection of the delta I-507 mutation in nine samples of human DNA that have been amplified by PCR.
Figure 6: HLM a~alysis of ~ three samples of human DNA
amplified by PCR for the presence of the normal, delta F-508, and delta I-507 alleles.
Figure 7: EILM analysis of three samples of human DNA
amplified by PCR using the simultaneous hybridization-ligation protocol with Ta~ DNA ligase for the pre8ence of the normal, delta F-508, and delta I-507 alleles .
Figure 8: HLM discrlm;r-~ti~n of the G542X and Normal alleles .

WO ~5/27078 r~
~ 21 86465 Figure 9: Simultaneous detection of delta F-508 and G542X
mutations by HLM.
Figure lO: Discrimination of normal, G551D, G551S, and Q552X sequences by HLM with 32P-G551D.NOR or 3lP-G551D.CF
using T4 DNA ligase with 200 mM NaCl.
Figure ll: HLM with 3lP-G551D.NOR and 3lP-G551D.CF using T4 DNA ligase with 600 mM NaCl.
Figure 12: Discrimination of normal, G551D, G551S, and Q552X sequences by HLM with 3lP-G551D. CF or 3lP-G551D.NOR
using Ta~ DNA ligase with 200 mM RCl.
Figure 13: Comparison of assays for G551D with Taq DNA
ligase under differe~t salt conditions.
Figure 14: The p53 model $or systematic evaluation of ligation specificity. The top 3equence is that of the target (region f lanking codon 175 of the p53 gene) . The probe sequences are on the bottom. The positions marked "X~ and ~'Y" were systematically varied with the four nucleotides .
Figure 15: Calculated per cent ligation for the delta F-508 assay of PCR amplified samples.
Figure 16: The correlation of per cent ligation calculated from HLM with the gellotype of PCR amplified human DNA. The error bar6 represent the 99~6 confidence interval .
Figure 17: Discrimination of the delta F-508 and delta I-WosSn7078 218~¢~ ss~ - ~
507 sequence6 with delta F-508 probes l:)y T4 DNA ligase as a function of NaCl concentration.
Figure 18: Figure 18 represents the discrimination of 5'(2) and 3'(2) miP~-trhl~c using T4 DNA Ligase and Taq - DNA Ligase, in terms of per cent ligation.
Figure 19: Figure 19 shows the 3' (1) mismatch discrimination for different base pairs, in terms of percent ligation. Data is shown for 2 different rnnr~ntrationg of T4 DNA Ligage (1 nM and 240 nM).
Figure 20: Figure 20 shows the 5 ' (1) m; P~ -trh discrimination for different base pairs, in terms of percent ligation. Data is shown for 2 different concentrations of T4 DNA Ligase (1 nM and 240 nM).
Detailed Description of the Invention The novel tPrhn;~ has been developed for gene probe analyses. This first step of DN~ analysis generally involves using an ampl;f;rAt;orl (e.g., PCR) technir~ue to multiply the sequence of interest. Obviously, if sufficient r~,uantity of the unknown sequence is present in the assay sample, amplification may not be needed.
Following the amplification proces8, a hybridization-ligation methodology (}~M) is used to confirm the identity of the amplification product. To help identify the gene sequence, an easily separable particle (for example, a magnetic particle) is used along with an i~l~nt;f;~hle moiety (for example, a luminescent marker such as an acridinium ester).
The initial part of the technique involves the use of WO 95/27078 ~ 't 2t 86465 an amplification procedure to multiply the sequence being investigated if insuf f icient quantity of the sequence to be; ~ nt; f ied is present . PCR techniques have been known for several years. For example, Saiki et al de8cribed an enzymatic amplification technique for 13-globulin genomic sequences by providing 2 oligonucleotide primers flanking the region to be amplified, annealing the primers to strands of denatured genomic DNA and .~-~t.-nll; n~ them with a DNA polymerase from E. coli or Thermus ac~uaticus and deoxyribonucleosidetriphosphates, and repeating cycles of denaturation, annealing and extension (Saiki et al (230 Science 1350, 1985); Saiki et al (239 Science 487, 1988) ) . Various amplification techniques have been developed recently (e.g., ligase chain reaction (LCR) and Q~ Replicase), and any of the amplification techniques can be used instead of PCR or in combination with one or more other amplification techniques. Furthermore, it is anticipated that other amplifying techniques will be developed. The exact technique used for amplification is immaterial to the invention herein, and those with knowledge in gene probe procedures are assumed to be f:lm;l;:lr with the overall techniques used therein and the reasons for prefering one technique over another. The critical fact is that the relatively small sample of DNA
is amplified so that the analytical technique used thereon is more sensitive than it normally would be.
Typically PCR techniques involve the procedure described above. There are many variations of this basic te~ lln;qn~ known to those with skill in the art, for example those described in PCR Protocols (eds. Innis, MA, Gelfand, DH, Sninsky, JJ, and White, TJ, Academic Press, 1990) . Details of one example of how a PCR reaction is run is described in detail in the examples herein.

WO 95/27078 P._ '0~ i Once an amplified sample is available, this material is analyzed by HLM. In HLM, the sequence which i8 complementary to some or all of the target sequence is incorporated into 2 or more probe6 which are reacted with the target oligonucleotide. Much of the discussion here relates to the use of only 2 probes, but more than 2 can also be used, as discussed below. One portion of the complementary sequence is attached to an insoluble material that can easily be separated from a reaction mixture. For example, a magnetic particle might be used.
Another possible material is a material that can be separated by centrifugation from the reaction mixture.
The second portion of the complementary sequence is attached to a material that can be detected by an analytical technique. For example, a ~pm~ m;n~F~cent material, such as an acridinium ester, might be used.
other examples are f luorophores or chromophores . These 2 complementary sequences are hybridized to the target sequence. The hybridization solution n~nt~;n~ 8alt, typically apprn~;r-t-~ly 500 to 700 mM NaCl, with , nn~ ~ntrationg of about 600 mM being most preferred. The hybridization is carried out at elevated temperature (e . g ., 45C . ) . Af ter hybridization, the insoluble properties of one of the probes is used to separate the hybridized from unhybridized probe with the label. A
ligase is then used to attempt to ioin the 2 complementary sequences of the probes. If the 2 complementary sequences precisely match the target in the immediate region of the junction of the two probes, the terminal nucleic acids are close enough to each other to be connected by the ligase.
On the other hand, if the target sequence is sufficiently different from the 8uspected target, the terminal nucleic acids on the probe8 are sufficiently far from each other so that they cannot be joined by the ligase. For example, ~ w09s~7078 2t~646~
if the target has a deleted nucleotide at the place where the 2 probes meet, the terminal nucleotide on one probe will overlap the other probe, and the two probes will not be ligated. Similarly, if the target has an inserted nucleotide, the 2 probes will not be close enough to each other to permit ligation to occur. Furthermore, if there is a mismatch at the location where the 2 probes meet, the 2 probes will not be ligated efficiently under conditions def ined herein .
Although the ligation proceeds as described above, depending on whether the tprm;ni~l bases of the probes couple or iail to couple with the target, those with skill in the art will recognize that the m;~m-trh;ng of bases on the probe away from the terminal positions may also have some effect on the binding of the probe to the target.
For example, if a base located on the probe several base positions away from the t~orm;n~l base fails to bind with the corresponding base on the target, there may not be sufficient discordance between the probe and target to prevent the ~rm;n~l probe bases from being ligated.
Steric factor and the totality of binding between the probe and target will have an effect on whether the instant technique is totally effective in determining the composition of the target. On the other hand, it should be noted that discordance away from the junction o~ the probes may have a sufficient effect 80 that the two probes will not be ligated. An example below shows a case in which discordance at a site 2 bases away from the junction of the probes causes sufficient interference that the probes were not ligated.
Similarly, by using sets of probes that are ~yp~rt~d to hybridize and ligate to a portion of the normal sequence, it i5 possible to determine whether the target rnntA;n~ a mutation. If it ig found that hybridization or ligation do not occur, it can be concluded that the mutation probably occurs iIl that portion of the target being f.~2m; n~d, By moving to the next portion of the target, a similar experiment can be run. Thus, by moving seql~nt;~1ly along the target, one can determine the site or sites on the target where mutations are found and then proceed to design experiments to identify the exact mutation which occurs at each of the mutation sites.
The ligation is carried out under conditions which will ensure the specificity of the reaction (see examples below) . ~igation can be carried out using one of the many 1 ;r;~t;nrJ reagents available, such ligating reagents typically achieving ligation by rhPm; rAl or enzymatic action~ One important difference between the conditions utilized here v8. the prior art is that a much higher salt rnnr~ntration hab been found to assure ligation specificity. The previously used salt concPnt~Atinn (200 mM NaCl) had been found to permit ligation of m; ~'tr~
probes. In the instant invention, it has been found that higher salt rrnr~ontrations ~yield unexpectedly; _ ~vt:d specificity. For example, to attain the most specific ligation with T4 DNA ligase, the salt rrnr~ntration is typically approximately 500 to 700 mM NaCl, with rnnrf-ntrationS of about 600 mM being most preferred and concentrations up to about 1000 mM being usable.
Variations in the ligation process are possible. For example, many different ligating agents can be used, and examples showing the use of T4 DNA-ligase and Taq DNA-ligase are shown herein. Furth~ ~, it may be preferred to include in the Taq ligase buffer other _ ^~t~ which ~ W0 95/27078 2 1 8 6 4 6 5 r~ c - c may increase the sensitivity of the reaction. For example, it has been found that the inclusion of tRNA
reduces background signal which is caused by non-specific bonding of the labeled probe.
After the ligation step is undertaken, a denaturation step separates the target sequence from that of the probes and ligated from unligated probe. The material connected to the insoluble material can then be separated from the reaction mixture by centrifugation, application of a magnetic field or other c-~Lu~Liate procedure, and the presence of any label connected to the insoluble material due to the action of the ligase can be determined.
By using the disclosed r~ ntFI~ the separation of the target material can be achieved using a technique other than C1ILI tography or electrophoresis. Thus the technique can be accomplished much faster than if electrophoresis or C1~L~ to~aphy were used. Furth, e, the detection technique can be a more quantitative one, such as the measurement of r~;o~ct;vity, fluorescence or luminescence. Other detection methods can utilize commonly available technique6 that permit the subsequent addition of the label. For example, the probe might have chemically attached thereto biotin, and, after separation of the probes, the label, being bonded to avidin or streptavidin, can be reacted thereto, thus forming a probe linked to a detectable label. Thirdly, the technique can now be utilized in some of the automated instruments, such as the ACS 180 instrument manufactured by Ciba Corning Diagnostics Corp. of Medfield, MA.
It should be noted that a variation of the instant technique can be used to determine information about the WOgS/27078 ?~ 6~ r~
sequence on the target. If, after denaturation, it is found that no label is connected to the insoluble probe, a different ali~[uot of the reaction mixture which has not been denatured can be further analyzed. In that sample, the ;n~Qlllhle marker can be separated (e.g., by the application of a magnetic field, by use of centrifugation, etc. ), and the target can be analyzed to determine if the marker probe is attached to the target. If this is found to be the case, the sequence of much of the target polynucleotide can be predicted due to the hybridization of the 2 probes to the target, and further experiments to confirm the se5[uence in that region of the target (e.g., near the ligation point ) can be planned .
Alternatively, a similar analysis can be cnnfl1~-t.otl by denaturing the sample after ligation and then separating the solid phase. In this technique, both the separated solid phase and the sllr~rn~t~nt are analyzed for the presence of the labeled probe. If most of the label is found only in the supernatant, it can be concluded that ligation of the probes did not occur, which is an indication of a mismatch at the expected ligation point.
~owever, since the labeled probe became attached to the target, it can be concluded that the target had the expected sequence, or a sequence close to expected, or else the labeled probe would not have hybridized to the target. Thus, even though ligation has not occurred, much can be inf erred about the sequence of the target .
Furth~ e, the sum of the amount8 of the label in the 8Up~n~t~nt and on the solid phase should approximate the total amount of target in the assay sample. The percentage of the labeled probe which is ligated should indicate the homozygosity or heterozygosity of the sample. In addition the ratio of label on the solid phase to the label in WO 95/27078 r~
~ 21 86465 solution can indicate more information a~out the se~uence on the probe, for example the existence of diseases wherein portions of genetic material are replicated (e.g., fragile X). It should be noted that, in conducting these assays, the data have not been found to be exactly the theoretically expected values (i.e., not 10096 of the label i8 found in the solid phase for a homozygous sample).
(See example below. ) Thus, by knowing the potential mutations that can occur at a particular site, it is possible to generate a specif ic probe 80 that the sequence on the target can be confirmed. If several potential mutations can occur at one site, it i8 also possible to design several probes, each with a different label, to determine whether it is the normal sequence, and, if it i8 a mutated 6e~auence, which of the mutations occurs. Similarly, mutations that occur near each other in a target sequence can be determined. ~In addition, the technique of using two or more differently labeled probes can be ut;l;7~ in the case where multiple forms of the target are ~ rt~l, such as in the fragile X case discussed above.
The two different labels usea in the same assay may be, for example, a fluorescent donor and fluorescent acceptor pair. In this case, by varying the ;nr;tlPnt light, the two labels can be used to distinguish among three possible outcomes . If the ; nri~nt light to the first probe gives fluorescence typical of the first label, this is an indication that only the first target is present . If the f luorescent output is that f rom the second label, two alternatives are present . If ; n~ nt light which excites the f irst label gives f luorescense from the second label, this is al~ indication that both W095/27078 21 8 b 4 65 ~
targets are present. On the other hand, if only the ;n~ nt light which excites the second label gives fluorescense from the second label, this is an indication that only target two is present.
For example, multiple mutations in one vicinity have been found in variations of cystic fibrosis. For example, the delta F-508 and delta I-507, tAt;nnc are both 3 base pair deletions, the positions of these mutations are partially overlapping in the sequence of the CFTR gene.
(See Zielenski et al (10 Genomics 214, 1991) for sequence of the CFTR gene.) Analysis of the PCR l;f;/-At;~n product ~rAnn; n~ this sequence by polyacrylamide gel electrophoresis would not be able to readily resolve these two mutations since the products would have the same size.
E~owever, these mutations would be distinguished by the ~M. In addition, exon 11 of the CFTR gene c~nt~;nfl the sites of many CF mutations including the G542X mutation at base 1756 (see SEQ ID NO 11) and G551D mutation at base 1784 (see SEQ ID NO 16) . After a single PCR amplification of the sequence spanning the sites of these mutations, the presence or absence of both mutations can readily be determined by HBM.
It will be noticed that, although there are some similarities between the HLM technique and ligase chain reaction (LCR), the techniques are, in actuality, very different. LCR itself is an amplification technique that has been known for some time. See, for example, Wu and Wallace, 4 Genomics 560, 1989; also Barany, 88 Proc.
Natl. Acad. Sci. USA 189, 1991. In LCR, two portions of oligonucleotides that are each complementary to each chain of a piece of the target gene being amplified (with the two together corr~p-~nrl ~ n~ to the entire target gene W09~/27078 2 1 8 6 4 6 5 r~l"D c ~ -portion) are added to the gene sample to be ;~mrl; f;
along with a ligase If the added oligonucleotides complement the target sequence, the ligase will j oin the two oligonucleotides . LCR is an ~3mrlif; c~tion technique wherein, knowing the sequence to be amplified, it is possible to add to the reaction mixture fragments that are complementary to the target so that, when ligase is added, ligation occurs and the target is amplif ied . LCR is ;nt~n~l~s3 to be repeated for several cycles so that large quantities of the desired sequence can be ~roduced.
~ILM, on the other hand, is an analytical technique, wherein probes to one or more suspected sequences which are likely to be found in the target are added to the target . The probes are j oined either to a material which can aid separation f rom the reaction mixture or to a label . Furthermore, the reaction is ; ntPnllP~l to be run f or only one cycle .
There are many variations possible in the EILM
procedure. For example, one may combine the detection procedure with column chromatography. One of the oligonucleotide probes in this procedure c~nt~;nf~ a substituent that will cause it to adhere to a column chromatograph. For example, one of the probes is biotinylated, and the ligated products are separated on avidin-sepharose. The other oligonucleotide might contain a fluorescent marker. Thus, when the sample is passed through a column chromatograph, those oligonucleotides that have been ligated will adhere to the chromatograph and be fluorescent. Thus fluorescence within the column is an indicator that the ligation has occurred. The use of the fluorescence donor/acceptor pair discussed above can also be used in the column chromatography technique.

W095/27078 P~I/1L c ~ -21 ~6~
Another variation deals with the moieties to which the probes are connected. Although in most cases one probe will be r-nnn~c~d to a moiety that permits separation of the ligated probes and the second probe will be attached to a label moiety, it is poss;hl-~ that the two probes can be attached to other moieties, such as other S=~ For example, the probes can be cnnnert~cl to the two Ls of the midivariant seyu~ e, if the QB
r~rl i o ~e system is being used. In this case, one probe is connPct~A to one portion of the midivariant sequence (e . g., midivariant A) and the second is connected to the second portion of midivariant sequence (i.e., midivariant B). If replication is observed in the reaction with QB
replicase after the ligation step, it is an indication that the two probes were ligated. The fact that the probes were ligated is an indication that the probec have the same sQT~nre as the target.
A further variation of the terhniql~ involves the timing for the addition of the flash reagent when using certain l~ inP~r~nt labels, such as acridinium esters.
After denaturation and separation of the ligated probes, it is possible to add DNAase before the addition of the flash reagent. This will allow for a more sensitive test, ~ince it has been found that the presence of the insoluble probe interferes at times with the amount o~ light given off when the flash reagents are added. The separation of the insoluble probe before the addition of the flash reagent thus allows a higher specific signal to be 3 o generated .
, Another variation ~ L~ the number of probes which are utilised. Although the preferred technique involves the use of two probes, one which aids in separation and W095/27078 21 ~6465 r ,, c one which aids in detection, it c-hould be noted that more than two probes can be used. In this case, one of the probes contains the separating moiety and one the detection moiety. If the separating moiety i8 on one tPrm;ns~l probe and the label moiety on the other ~Prm;n~l probe, the probes between them would not need to contain a label. In this experiment, if, after ligation, the label probe i8 attached to the separating probe, it can be conc-lll~P~ that the int~ te probes also habridized to the target, for, if this were not the case, the label probe would not be ligated to the probe moiety which i nr~ PC the separating probe . Another variation of the multiple probe experiment is the one where one probe cont:~; nPd the separating moiety and all the other probes are labeled. In this case, after ligation, the amount of label attached to the separated moiety is an indication of whether all the probes were ligated. The use of more than 2 probes, when ~;nDd with~ the variations ~iccllc~e~3 above (e.g., analysis with and without ligation, analysis before d~ Lu ~tion, etc. ~ leads to a number of analytical variations which will be apparent to those with ordinary skill in this area.
Fur~h~ ~, the location of the separating or ~Ptect;ng moiety can be varied. I~ hn~h it is preferred that they be at the ~Prmin~l end of a probe, it is poRsihlP that they be connected to the probe in any location so long as they do not interfere with hybridization and ligation.
A further advantage of the novel technique is that it is now possihlp to descriminate between two mutations that are closely related to each other te . g ., mutations that occur at adjacent nucleotides).

A further adva~tage of the instant invention is that differe~Lt markers can be used on different prQbes in the same experiment. Thus, for example, the presence of one of two possible mutatiQns can be det~nn;n~rl in one test, with each probe using a different marker Both markers could be separated from the sample and the two could be distinguished by their differing absorption spectra, for example. Fur~ , by using different insoluble particles, the 2 probes can be separated from each other before analysis. For example, if one probe relies on an insolu~le non-magnetic particle and the other uses an insoluble magnetic particle, the magnetic field could be applied f irst in order to remove the magnetic particles and the markers attached thereto, and the Ll ; n; nr~
solution can be centrifuged to remove the non-magnetic insoluble particles with its attached marker. Ot~er variations of these separatior~ techniques will be apparent to those with skill in the art. Thus, these two markers could still be distinguished f rom each other even though they both have the same label. By combining in one experiment variations of techni~ues 1lt;l;7~hle for both separation and detection, it is po~sible to determine the presence of one of several mutations or other genetic variations in the one experiment. For example, magnetic (M) particles could be used on some prQbes, non-magnetic (NM) on others; some of the second oligonucleotides i~the probe could use r~ n; l nf~cent material A, while others could use chemiluminegcent material B. Thus, by varying just these 2 parameters, 4 mutations could be detected in one experiment. (I.e., where the adducts to the probes are M-A, NM-A, M-B, NM-B) By lltil;7;nr~ particles that can be separated from each other with different markers (i.e., those with different spectral or other characteristics), it would be possible to detect many genetic variations in one experiment.
A further advantage of the novel procedure is that the technique is more sensitive than previous techniques.
This increased sensitivity is due to several factors. For example, the analytical techniques for ~l~t~rm;n;ng the presence of target material are more sensitive; the solution technique of separating the insoluble particle and analyzing the marker attached thereto is much more sensitive than the process of using electrophoresis to separate the components and to rely on staining to qualitatively and quantitatively determine the presence of the target .
Further varlations of the above procedure are possible. For example, after the insoluble particles are separated f rom the reaction mixture, the quantity of target present can be detPrm; n-~d by measuring the amount of marker while the marker is still attached to the insoluble material when the insoluble particles are precipitated (as, for example, using a classical quantitative analysis on insoluble material).
Alternatively, after the insoluble particles are separated from the reaction mixture, the particles can be 2~ resuspended and the marker determined while on the r~sl~pPnf~ particle. Furthermore, the marker can be separated from the insoluble particle and be measured when both the marker and insoluble particle are in solution or suspended. Also, after separation of the marker from the insoluble particle, the insoluble particle can be separated from the solution and the marker can be measured in the absence of the insoluble particle.
n addition to detecting specific DNA sequences, W095/27078 r~l, S~

may also be used to detect specific RNA sPS[n~nrP~ provided that the ligase used can ligate probes which are hybridized to an RNA target. The terhn;que can also be used to analyze viral materials and other se,tu~ances of polynucleic acids. In addition, since the examples illustrate the ability of HLM to distinguish sç~ c which differ at positions nt sites other than the ligation ~unction of the probes, this method can be readily adapted to scan large B-, ts of sequence even up to whole genes for alterations distinct from a normal sequence.
Further variations of the invention will be apparent to those with ordinary skill in the art. The following 1PC illu8trate varioug a8pectg of the invention but are not intended to limit its usefulness.
Example 1: Simultaneous Detection of Normal and Delta F--508 Alleles Using 'hPmi 1 jnpcr-pnt Hybridization Ligation Assay with DMAE and 2 0 LEAE Labeled Probes A chemill~m;nPccPnt hybridization-ligation method was tested for it6 ability to detect the delta F-508 (SEQ ID
N0 1) mutation in cystic fibrosis in samples of human DNA
amplified by PCR. In addition, the l~L_Se~l- e of both the normal tSEQ ID NO 2 ) and delta F-508 alleles was simultaneously determined for each sample by using probes specific for each allele but labeled with two different acridinium ester derivatives (DMAE and LEAE). The DMAE
derivative (dimethyl acridinium ester) rhPmilllminP~rP~ at the shorter wavelength range (400-500 nm) and LEAE (longer wavelength emitting acridinium ester) chemilllminPccPc at the longer wavelength range (500-600 nm).

WO 9~l27078 2 1 8 6 4 6 5 r~
Nine samples of human DNA (250 ng) obtained from an outside laboratory were amplified by the polymerase chain reaction (~3ee Saiki et al, 239 Science 487, 1988). The primers used were obtained from Genset (Paris, France) and had the f ollowing seguences:
C16B (Seguence ID NO 3): 5' GTT TTC CTG GAT TAT GCC TGG
CAC 3 ' C16D (Seguence ID NO 4): 5' GTT GGC ATG CTT TGA TGA CGC
TTC 3 ' The target seguence amplified in the PCR reaction with these primers consisted of 97 bp (94 bp for delta F-508 allele) spanning bases 1611-1708 of the CFTR gene (3) .
The PCR r~ct;nn~ (75 ul) ~mt~in~od 30 pmol each primer, 1.9 mM MgCl~, 200 uM each ATP, TTP, GTP, and CTP;
and 2.5 U Tag DNA polymerase. After denaturation at 95C
for 5 min,the samples were amplified by 30 cycles of PCR
consisting of isnn~l ;n~ at 60C for 45 sec, extension at 72C for 1 min and denaturation at 95C for 45 sec. After the last cycle the samples were incubated at 72C for 5 min .
An aliguot of the PCR reaction solution was denatured and then added to the chemiluminescent detection reaction:
100 ul TE, 4X SSC, 0.19~ BSA, 0.029~ Tween-20, 59~ dextran sulfate c~ nt~;n;n~ 10 ug paramagnetic particles (PMP) with the immobilized probe (PMP) and 100 fmol of each acridinium ester (508.CF-DMAE and 508.NOR-I-EAE) labeled probe. The seguences of the detection probes were:

WO 95/27078 , I _ ' . ~

PMP.508 (SEQ ID NO 5): 5' CCT AGT CCA AGT ACG GCG CCG AAG
AGG CCC TAT ATT CAT CAT AGG AAA CAC CA 3 ' 508.CF (SEQ ID NO 6): 5' ATG ATA TTT TCT TTA ATG GTG CCA
3' 508.NOR (SEQ ID NO 7): 5' AAG ATG ATA TTT TCT TTA ATG GTG
CCA 3 ' The possible assay formats are summarized as shown in Figure 1.
The probes were hybridized to the PCR product at 45C
for 15 minutes. Unhybridized AE probes were removed by magnetic separation of the particles and decanting the SUP~:L..at,1.1~. The particles were washed with 2X SSC/0. 1%
Tween-2 0 .
Hybridized probes were ligated by l _~ i n~
20particles in 100 ul 50 mM Tris, pH 7.6, 10 mM MgCl2, 1 mM
ATP, 1 mM DTT, 5% polyethelyene glycol 8000 and 200 mM
NaCl containing 2 U T4 DNA ligase. Reactions were incubated at 37C for 15 minute6. After separating and washing the particles as described, the hybridized but non-ligated AE probes were ~lRcociAted by ~ lin~ the particles in 150 ul H20 and incubating at 65C for 10 minutes. The particles were separated and the Du~L.,a~unt containing ~li eeoci Ated AE probe removed. The particles were washed once as described and then ~ 1 in 100 ul 10 mM Tris, pH 7.5, 10 mM MgCl2, 1 mM EDTA, and 0.05 ug/ul DNase I (8RL). The particle ~lep~-nRi~n wa6 flashed using standard flash reagents (see, for example, Law et ~ WO 9~127078 2 1 ~ 6 4 6 5 }~I/~ t~
al, U.S. Patent No. 5,241,070) and the chemiluminescence detected in a dual wavelength 1~ tt~ 80 that the chemiluminescent signalE from the two different labels were measured simultaneously.
The results of the chemiluminescent hybritl;7atinn-ligation assay for PCR amplified product are 6hown in Figure 2. The chemiluminescent signal obtained clearly ;t~t~nt;f;t~tl the pregence of the delta F-508 and normal alleles in the PCR amplified products. As expected, the allele specific probes hybridized to each sample irrespective of genotype (data not shown), the subsetluent ligation step discriminated between the se~auences of the two different alleles since efficient ligation was observed only at the junction of the hybridized probes which were perfectly complementary with the target setluence (see Figure 1) .
The chemiluminéscent detecti on of these alleles allowed the diagnosis of these samples which was in complete accord with the analysis of the same samples by an independent laboratory with one exception (See Example 2) .
~ach sample was analyzed with both delta F-508 and normal allele specific AE probes in order to unambiguously assign the genotype of the samples. The magnitude of the chemiluminescence (Figure 2) also indicated the genotype of the samplç in that an int~ te level of chemiluminescence was observed for heteL~,zy~uus individuals (for example the normal allele in samples 1,7, and 9) relative to the chemiluminescence of the ht -l~y~L~us cases. Sample 7 was an exceptional sample in that the magnitude of the chemiluminescence from the 508.NOR-LEAE

Wo 95/27078 2 1 ~ 6 4 6 5 probe indicated that the sample was heterozygote for this allele, but the signal from the 508 . CF-DMAE probe indicated that this sample was negative for the delta F-508 mutation. Taken together these results suggested that the second allele in thi8 sample cnnt~in~A neither the delta F-508 nor the normal sequence but a second cystic fibrosis mutation instead. These PCR products were al80 analyzed by electrophoresi8 on an 8M urea/1096 polyacrylamide gel and the resolved bands visualized by ethidium bromide staining (data not shown).
The products from the normal (97 bp) and CF (94 bp) alleles were clearly resolved on the gel, ~n~hl; n~ a nnRi R . In addition, heterozygote samples also cnnt~;n~d bands resulting from heteroduplex formation which migrated as ~t,al~:L,Lly larger products. In this regard, on the basis of the electrophoretic analysis, samples 1, 7, and 9 appeared identical and would be assigned a8 delta F-508/normal heterozygotes. As noted above, on the ba8is of the chemilumine8cent data, although sample 7 was het~ ,;y~uus for the normal allele, it did not appear to contain the delta F-508 allele. This discrepancy was resolved in Example 2 and illustrated the ability of I~LM to provide a more accurate diagnosis , -- ed with standard analytical procedures such a8 electrophoresis.
Example 2: Discrimination of Delta F-508 and Delta I-507 Mutations by Chemil~lm;nRRcRnt Hybri~1;7z~t;nn 3 0 ~igation The delta F-508 mutation is the most common mutation in cystic fibrosis (CF), occurring in approximately 68% of the cystic fibro8is chrmomosomes (3). This mutation is a WO 95J2N78 1~ C '~ -21 ~6465 deletion of three base pairs in exon 10 of the CFTR gene (3). The sequence of exon 10 ~uLL~ullding this mutation site is shown in Figure 3. The delta I-507 (SEQ ID N0 8) mutation is a much rarer CF mutation which is also a three base pair deletion that partially overlAps the delta F-508 mutation (Figure 3). The sequences of these two CF
alleles differ by a single base.
The ability of the ~h~m;lllm~nacc~nt hybridization ligation assay to distinguish the delta F-508 and normal alleles was d ~L~Ited in Example 1 . The ability of the assay to dist;n~ich the delta F-508 and delta I-507 alleles was shown in this e~ample. This application requires that the ligation step distinguish s~l ~r,- ac which differ at a single position. In addition, the site of the mismatch in these hybrids occurs at one base removed from the ligation junction. The hybrids formed between the delta F-508 and delta I-507 probe sets and the different target sa-lu~ aC are shown in Figure 4.
The same nine ~l ini~ Al samples of human DNA which had been amplified by the polymerase chain reaction (example 1) were analyzed for the ~L~E_., e of the delta I-507 allele. In this assay, a similar format was followed as in example 1 with the exception that the solid phase and acridinium ester (DMAE) labeled probes were as follows:
PMP.507 (SEQ ID N0 9): 5' CCT AGT CCA AGT ACG GCG CCG
AAG AGG CCC TAT ATT CAT CAT AGG AAA CAC CAA AG 3 ' 507.CF (SEQ ID NO 10): 5' ATA TTT TCT TTA ATG GTG CCA GGC
3' An additional three samples (30, 31, and 32) of PCR

WO 95/27078 r~ L _ ~ r - ~

amplified human DNA were assayed for the delta F-508, delta I-507, and normal alleles using the same protocol as well as a protocol ~lt;l;7;ns= Taq DNA ligase (Epicentre Technologies) . The thermostablility of Taq DNA ligase permitted the ligation reaction to be carried out at higher temperature as well as allowing the hybr~ 7~t;on and ligation steps to be carried out simultaneously. The buffer for the simultaneoug hybri~i;7at;on-ligation consisted of 20 mM Tris, pX 8.3, 200 mM RCl, 10 uM tRNA, 10 mM MgCl2, 0.5 mM NAD, and 0,0196 Triton X-100. The r~ ct;l~nc cf)nt~in~-1 100 units Taq DNA ligase. The simultaneous hybridization-ligations were carried out at 60C for 30 minutes. The ,~ ;n~f~r of the protocol was i~nt;c~l to that for the assays employing T4 DNA ligase.
The results of the l-~hem; lllm;n~lcent hybriri; 7~t; ~n ligation assay of the nine samples are shown in Figure 5.
Only sample 7 was positive for the delta I-507 allele.
These results taken together with those of Example permit the following assignments for these samples:
Table I
(i~;N~'l'Yl~ OF CLINICAL SAMPLES
1 ~ ~ 4 e; ~ 7 R 4 The results of the three additional samples assayed for the delta F-508, delta I-507, and normal alleles are shown in Figures 6 and 7. The assignments made for these samples were cr~nf; -~1 by sequencing.
The results shown in examples 1 and 2 illustrate the W0 95127078 r~
21 ~6465 ability of chemiluminescent hybridization-ligation assay to discriminate sequences differing by a single base even when the 8ite of this difference occurs one base removed from the site of the 1 1 gPt; ~n junctioll . In this particular application, the cystic fibrosis mutations delta F-508 and delta I-507 were di8tinguished. This enabled samples that had been previously characterized as delta F-508/N to be correctly assigned as delta I-507/N. The assignment of samples 7 and 30 as delta F-508/~ heterozygotes had been made on the basis of the electrophoretic mobility of the PCR products of these samples. But analysis of these PCR
products by polyacrylamide gel electrophoresis fails to readily distinguish the delta F-508 and delta I-507 mutations, since both mutations consist of three base pair deletions, the PCR products from these alleles are the same size .
Example 3: Assay for the G542X Cystic Fibrosis Mutation The previous examples have demonstrated the ability of the chemiluminescent hybridization-ligation assay to distinguiF3h the normal and cystic fibrosis alleles at the sites of the delta ~-508 and delta I-507 mutations. A third cystic fibrosis mutation, G542X (SEQ ID NO 11), is a point mutation occurring in exon 11 of the CFTR gene. The current method f or detecting this mutation requires sequencing, which is a lengthy and laborious procedure.
The chemiluminescent hybridization-ligation assay to detect this , ~,~t; ~n must be able to distinguish the single base substitution which differs between the normal (SEQ ID NO
12) and cy8tic fibrosis alleles.
The same simultaneous hybridization-ligation assay using Taq DNA ligase as described in Example 2 was used in W095/27078 2186~65 ~ --the G542X assay. The g~-lu-. o~ of the G542X probes were:
PMP.G542X tSEQ ID NO 13): 5' CCT AGT CCA AGT ACG GCG CCG
AAG AGG CCA CTC AGT GTG ATT CCA CCT TCT C 3 ' 5G542X.CF (SEQ ID NO 14): 5' AAA GAA CTA TAT TGT CTT TCT
CTG CAA 3 ' G542X.NOR tSEQ ID NO 15): 5'CAA GAA CTA TAT TGT CTT TCT
CTG CAA 3 ' The results of the assay are shown in Figure 8 and indicate that the only G542X.CF probe was ligated with the G542X
sequence and only the G542X.NOR probe was ligated with the normal sequence. In thi6 example, T-C and G-A mismatches at the ligation junction were not efficiently ligated.
Example 4: Simultaneous Assay for Delta F-508 and G542X
The ~h~ ;n~c~nt hybridization-ligation assay can be used for the simultaneous detection of multiple sequences. One illustration of this rAr~-h; 1 ;ty was the simultaneous detection of the delta F-508 and normal alleles in a single assay (Example 1). Another application is the detection of two or more mutations which underly an inherited disease or cancer. For example, more than 200 mutations have been described which underly cystic fibrosis. The delta F-508 mutation is the most common one, occurring in approYimately 68% of the cystic fibrosis The second most ~L.~ue~.l cystic fibrosis mutation is the G542X mutation, a point mutation occurring in exon 11 of the CFTR gene. Instead of using the two different acridinium ester labels to simultaneously detect the normal and CF alleles at a single locus, the delta F-~ wo gs/27078 6 5 r~ c ~ ~ -508 and G542X mutations may be detectd in a single assay.
In principle, as many mutations a~ there are acridinium ester derivatives with distinct ~hf~m; 1 l~m; n~cent properties, may be detected in a single assay. Alternative labels, such as fluorophores, may permit a still greater number of loci to be simultaneously detected.
A model assay to test feasibility was performed with synthetic target sequences. The assay protocol was the same as that described in Bxample 2 using Taq DNA ligase and simultaneous hybridization-ligation. Two solid phases and two acridinium ester labeled probes, one set each for the delta F-508 and G542X mutations were used. The probes for the the delta F-508 mllt~t;r~n were the same as those described in Example 1 with the exception that the acridinium ester label was LEAE. The sequences of the G542X probes were the same as those used in example 3. The results of the assay are summarized in Figure 9 and show that the delta F-508 and G542X sequences were (~f~tf~t~fl with whatever combinations of targets were employed. This demonstrates the f~R;h;l;ty of detecting multiple genetic mutations in the same assay.
Example 5: Discrimination of G551D, G551S, and Q552X
Cystic Fibrosis Mutations.
Exon 11 of~ the CFTR gene cnnt~;n~ the sites for many other cystic f ibrosis mutations in addition to the G542X
mutation described in the examples above. The presence of multiple mutation sites in the relatively short span of sequence of exon 11 has heretofore resulted in the necessity of sequencing the PCR product from this exon in order to detect and discriminate these possible mutations.

WO 95/27078 ~ 5 The ability of H~ to simplify analysis of eYon 11 cystic fibrosis mutations required that the specificity of this method enable discrimination of closely clustered mutation sites. The G551D mutation (SEQ ID NO 16) i5 one of the more common cystic fibrosis mutations, accounting for approximately 0 . 5% of the observed frequency, this is a point mutation in which G1784 in the normal gene tSEQ ID
NO 17) is changed into an A. close to the site of the G551D are the G551S (SEQ ID NO 18) at base 1783, and Q552X
(SEQ ID NO 19) at base 178 . In addition the nature of the G551D mutation requires the discrimination of a G-T
mismatch by HLM, one of the most dif f icult mismatches to discriminate (see below). The ability of HLM to detect the G551D mutation and discriminate between it and the other mutation sites near to it was ~ LLc.ted in this le .
The E~ "n~ of the probes used in the G551D assay were as follows:
PMP.G551D (SEQ ID NO 20): CCT AGT CCA AGT ACG GCG CCG AAG
AGG CCC TAA AGA AAT TCT TGC TCG TTG A
G551D.CF (SEQ ID NO 21): TC TCC ACT CAG TGT GAT TCC AC
G551D.NOR (SEQ ID NO 22): CC TCC ACT CAG TGT GAT TCC AC
In these assays, G551D.CF and G551D.NOR were labeled with ~2p at their 5' termini. Detection of ligation product was accomplished by liquid scintillation counting. Assays were performed using either the standard T4 DNA ligase and Taq DNA ligase protocols described above as well as modif ications to these protocols by altering the salt .

W0 95/27078 l ~
2~ 65 conditions in order to improve specif icity of HLM . For T4 DNA ligase, this involved increasing the NaCl ~nn~-~ntration from 200 to 600 mM. The Taq DNA ligase protocol was altered by substituting NaCl f or KCl . The exact ligation s conditions are indicated with the f igures below.
The results of HLM analysis using the T4 DNA ligase protocol with 200 m.M NaCl are shown in Figure 10. Although HLM discr; m; n~ted between the G551D sequence and the sequences for the other I ~tion~, there was essentially no discrimination between the G551D sequence and the normal sequence with the G551D. CF probe. The 3pecifity for HLM
using T4 DNA ligase was improved by increasing the NaCl concentration to 600 mM (Figure 11). Even better discrimination was obtained by employing HLM with Taq DNA
ligase (Figure 12), under these conditions the different sequences were readily disrr;m1n~ted with either G551D.CF
or G551D.NOR probes. Using the the Taq DNA ligase protocol with 200 mM KCl and G551D.CF, discr;m1n~t;nn was readily apparent between the G551D and normal sequences, but some ligation above background was observed with the normal target. This signal was able to be depressed further by substitution of NaCl into the Taq DNA ligase protocol (Figure 13).
Taken to~eth~r these results indicate the ability to HLM to discriminate sequences which differ by only a single base, even when the site of the single base change occurs at positions removed from the ligation junction.
Example 6: Systematic Evaluation of the Spe--l f; ~; ty of Hybridization-Ligation Ut; l; 7; n~ a p53 Model .
The preceding examples have establish the ability of Wo 95/2N78 2 1 8 6 ~ 6 5 r~
HLM to discriminate sequences with subtle differences including deletions, insertions, and point mutations. In each of the cases P~r~m;n/~rl conditions were established which permitted the specificity of ligation to discriminate these sequence differences. In this example, the specificity of H~M is systematically evaluated by testing its ability to discriminate se~tauences which differ by all of the possible combinations of mismatche8.
The p53 gene codes for a protein which functions as a tumor suppressor. Mutations in thi8 gene are observed in a wide variety of tumors, the most frequent positons for these ~ At i nn~ cluster about codons 175, 245, and 248 . A
portion of the p53 gene surrounding codon 175 (SEQ ID NO
23~ was chosen as the model for the systematic evaluation of the specif icity of ligation . The target and probe sequences for this model are shown in Figure 14.
Hybridization and ligation8 were carried out in solution.
For assays ut;li7;n~ T4 DNA ligase, probes (3~P-SEQ ID NO
24 and 25) were mixed with targets in ligation buffer rnnt:l;n;n~ the ;n~;c~te~l NaCl ~n~ntration~ Reactions were incubated at 37C for 15 min, T4 DNA ligase (1 U) added and reactions incubated at 37C for a further 15 min.
An aliquot of the reactions was analyzed by denaturing (8 M urea) polyacrylamide (15~) gel electrophoresis. Bands corr~pnnrl; n~ to the ligation product and the unligated oligomer were excised and counted by li~uid sc;nt;ll;~t;nn counting, and the per cent of the total oligomer ligated calculated. The as8ay8 utilizing Tasl DNA ligase were carried out similarly except that the ligase was added at the start of the r~ct;nns~ ~
The ability of T4 DNA ligase to discriminate mismatches occurring at the 5 ' pho8phate or 3 ' OH

~ W095/27078 21~6465 r ,,~
nucleotide at the ligation junction in 200 mM NaCl i8 summarized in Table II. Mismatches at the 3' OH nucleotide were much more easily discriminated than mismatches at the 5'P nucleotide. Increasing the NaCl concentration to 600 mM ~ ,v~d the specificity of T4 DNA ligase over that observed at 200 mM. At 600 mM NaCl, all possible mismatches at either the ~ ' P or 3 ' OH positions were discriminated, even the G-T and C-A mismatches (Table III) .
The specificity of Taq DNA ligase at 45C in 200 mM
RCl was better than that of T4 DNA ligase (Table IV).
Inclusion of NaCl in the Taq ligase buffer also improved the specificity of Taq DNA ligase somewhat (Table V) .

W0 95l27078 r~
- 21 ~`6465 TABLE II
SPECIFICITY OF T4 DNA LIGASE IN 200 mM NaCl~
TARGET BASE 3-~ O~I PROBE BASE 5 ~ P PROBE BASE
S
C T A G ¦ C T A G
10 G 80 51 1. 9 9 . 8 ¦ 52 41 15 31 A 20 72 1.4 3.6 ¦ 58 65 45 15 T 2 . 2 4 . 2 77 8 . 7 ¦ 56 54 62 64 C 1 . 4 11 53 69 1 10 21 29 40 ~The results summarized in~the table represent the percentages of the total amount of the limiting~probe which was ligated as determined by PAGE a~alysis. The table is arranged so that the complementary pairs of nucleotides fall on the diagonal, the off-diagonal entries are the pos~ible comb-n~tion~ of mismatches. Refer to Figure 14 for the sequence of the hybridis formed between probes and targets .

W095/27078 r_~,.. c TA~3LE I I I
SPECIFICITY OF T4 DNA LIGASE IN 600 mM NaCl~
TARGET BASE 3 ' OH PROBE BASE 5 ' P PROBE BASE

C T A G C T A G
10 G 54 1.1 0.7 1.5 ¦ 36 17 1.4 0.7 A 2.2 64 1.0 3.6 ¦ 3.0 31 1.3 1.0 T 1.1 1.3 55 2.0 ¦ 1.6 14 43 37 C 1.1 1.1 1.1 44 1 0.6 2.0 2.5 25 ^The results summarized in the table represent the percentages of the total amount of the limiting probe which was ligated as determined by PAGE analysis. The table is arranged so that the complementary pairs of nucleotides fall o~ the diagonal, the of ~-diagonal entries are the possible ' ;nA~ n~ of mismatches. Refer to Figure 14 for the sequence of the hybridis formed between probes and targets .

W0 95127078 -2 1 8 6 4 6 5 r~ c - - ~
TABI,E IV
SPECIFICITY OF Taq DNA I-IGASE IN 200 I:M KCl-TARGET BASE 3 l OH PROBE BASE 5 ' P PROBE BASE
C T A G C T A G
G 60 8.2 0.6 1.8 ¦ 34 14 0.6 1.9 15 A 0.8 64 1.1 2.6 ¦ 7.0 43 5.0 3.0 T 1. 5 2 . 7 71 3 . 3 ¦ 5 . 0 16 43 11 C 2.7 2.0 1.6 61 1 2.0 4.0 11 34 'The results summarized in the table represent the percentages of the total amount of the limiting probe which was ligated as determined by PAGE analysis. The table is arranged 80 that the complementary pairs of ~ucleotides fall on the diagonal, the off~ nn~l entries are the possible combinations of mlsmatches. Refer to Figure 14 for the sequence of the hybridis formed between probes and targets .

~ W095127078 21~36465 r ~
TABI.E V
SPECIFICITY OF Taq DNA LIGASE IN 25 mM KCl/75 mM NaCl~
TARGET BASE : 3 ' OH PROBE BASE
C T - A G
G 31 0.6 0.7 0.7 A 1.5 46 0.7 2.5 T 0.8 2.5 51 1.7 C 0.8 0.8 0.8 39 ~The results summarized in the table represent the percentages of the total amount of the limiting probe which was ligated as determined by PAGE analysis. The table is arranged 80 that the complementary pairs of nucleotides fall on the diagonal, the off-diagonal entries are the possible, ' n~tions of mismatches . Refer to Figure 14 for the sequence of the hybridis formed between probes and targets .

Wo 9~/27078 Example 7: Assays for the delta F-508 and Normal Alleles in PCR Amplified Human DNA Using Percent ~igation as Diagnostic Criterion Samples of PCR amplified human DNA were received from an independent laboratory. HLM analysis of the delta F-508 (SEQ ID NO 1) and normal (SEQ ID NO 2) alleles was perf ormed as described in the previous examples . At the denaturation step to remove the hybridized but unligated probe, the sl~rprnAtAnt rnn~Ain;ng this released probe was reserved and flashed separately, in additio~z to flAqh;
the PMP which rr~ntA;npd the hybridized and ligated labeled probe. The sum of the chemil7lm;nP~Pnt signals from the supernatant and PMP provides a measure of the total amount of labeled probe which hybridized to the sample. This in turn provides a measure of the total amount of sample DNA
in the reaction. In addition, the chemiluminescent signal from the PMP divided by the. sum of the chemiluminescent signals from the supernatant and PMP provides a measure of the fraction of the hybridized probe which was lir~ated.
Since the amount of PCR product obtained from each sample of human DNA may vary from sample to sample, the fraction of labeled probe ligated provides a clearer distinction between samples which are homozygous for an allele, samples which are heterozygous for an allele, and those which do not contain the allele. In addition, since the probes may be P~rPrt~-l to hybridize to delta F-508, normal, and delta I-507 alleles, determining the chemiluminescence of the labeled probe released at the denaturation step, provides a means to lPtPrm;nP that the assay c ~ ^n~ are functioning proeerly for those samples which are ligation negative The H~M results for the delta F-508 assay calculated ~ W09S/27078 21 ~ 6465 r~ s~l -as per cent ligation are summarized in Figure 15. A clear di6crimination between h( _yy~ls, heterozygous, and negative samples could be made based upon the calculated percentage of the labeled probe ligated. This calculated parameter turns out to be a more reliable diagnostic index than the raw chemiluminescent data because the samples were found to vary more than S-fold in the total amount of DNA
present (data not shown). In Figure 16, similar ~IDM
results for the assay for the normal allele are compared with those of the delta E~-508 assay.
Example 8: Effect of NaCl t~n~ntration on the Discrimination of Delta F-508 and Delta I-507 Alleles The sequences for these two alleles differ at a single position. (See Figure 4). Using XT,M with T4 DNA ligase in 200 mM NaCl, it was possible to discriminate between these two cystic $ibrosis mutations (See Examples 1 and 2). In light of the results in Example 6 showing the effect of NaCl rnn~ntration of the specificity of T4 DNA ligase with the p53 sequences, the effect of NaCl ~ ntration on the ability of the T4 DNA ligase to discriminate between the delta F-508 and delta I-507 sequences using the delta F-508 probes was Py~m;n~ ILM was performed on delta F-508 and delta I-507 synthetic target se~uellces as described except that ligation buffers were made up with either 200, 400, 600, 800, or 1000 m,M NaCl. ~t the denaturation step, the supernatant was reserved and flashed separately in order to determine the amount of hybridized but unligated probe.
The percentage of the 508 . CF-DMAE probe which had been ligated out of the total amount hybridized was calculated as described in Example 7.

Wo ~5/27078 r~ c - ~
2 ~ 8b4~5 The results are summarized in Figure 17. There was sufficient dlscrimination by T4 DNA ligase in 200 mM NaCl to distinguish between the delta F-508 and delta I-507 sequences. Increasing the salt concentration up to 600 mM
improved the discrim~n~tinn between these sequences by suppressing the amount of ligation observed with the delta I-507 sequence wkile the ~s~lnt~;nln~ the level of ligation with the delta F-508 sequence. At NaCl concentrations above 600 mM, the ligation with the delta F-508 target begins to decline.
Example 9. Hybridization-Ligation Assay for :~E-508 with Biotinylated Probe and Avidin-PMP.
Xybridization and ligation reactions were carried out insolution with a biotinylated probe ~Biotin-CF1) and an acridinium ester labeled probe (508.CF-}~).
Xybridization-ligation reactions were carried out in 100 ~Ll buf~er (20 mM Tris, pH 8.3, 100 mM NaCl, 100 mM KCl, 10 mM MgCl2, 10 ~M t RNA, 0.5 mM NAD and 0.01~ Triton X-100) containing 1 pmol Biotin-CF1, 100 fmol 508.CF-AE, and 100 units Taq DNA ligase at 50C~ Reactions were initiated by the addition of either 1) 1 fmol normal target, 2) l fmol ~I-507 target, 3) 1 fmol ~F-508 target or 4) No target. Reactions were incubated at 50C for 1 hour. To each reaction was added 10 ug avidin-PMP (Promega) and 1 nrl1h~tP~ an additional 10 minutes. The particles were separated magnetically, the g11rPrn~t~nt aspirated, and the particles washed with 0 . 2X SSC/0 .1~6 Tween-20 . The washes were repeated twice .
The avidin-PMP were rP~llRppn~ in 150 ~l wash buffer and incubated at 55C for 10 minutes to remove hybridized but unligated probe. The avidin-PMP were ~eparated magnetically, the supernatant removed and .

WO 95/27078 r~
21 86~65 flashed. The avidin-PMP were washed once, resuspened in 100 ~Ll 10 mM MgCl2, 50 mM Tris, pH 7.5 rrnt~;n;nrJ DNase I (BRL) and flashed. The results of the assay are summarized in the table below.
Tar~et Percent I,iqation Normal 0~
~I-507 249c ~F-508 609~
This assay format shows the ff~R; h; l; ty of the use of a universal solid phase reagent (avidin-PMP) with a biotinylated probe . This f ormat may be especially useful in multiplexing schemes where it is expected that assays for a number of genetic loci may be performed in the same tube. In addition, solution phase hybri~; 7~tirlnR and ligations may proceed more rapidly than those involving one probe immobilized on PMP.
~xample lO: ~ Ligation Specif icity and G551D Model Results These results extend those given in ~xample 5 in the instant application. Hybridization-ligation assays were carried out with T4 DNA and Taq DNA ligases as described previously. The results of these assays using the AE probes specific for the G551D sequence and the normal sequence are summarized in the table. Inclusion of the target sequence designated R553X provides additional insight into ligation sper; ~; rity (See WO95/27078 21 8 64 65 r~
Table VI . ) In the assay with the G551D.NOR probe, the G551S
and Q552X sequences were discriminated. Thi~
illustrates the ability of T4 and Taq DNA ligases to discriminate mismatches at positions other than at the ligation junction. Moreover, in the assay with G551D.NOR, T4 DNA ligase appears to be able to discriminate the R553X aequer~ce. This requires the enzyme to discriminate a mismatch 5 bases from the 3' hydroxyl side of the l;~t;nn junction.
The results of this assay illustrate the impLuv~ t in discrimination one can obtain when two m; qm~trh~q are present as opposed to a single m; ~ trh For example, in the assay with G551D.NOR, the R553X
sequence is not discriminated by Ta~ ligase. Viewed in a different way, the 3' (5) T-G mismatch does not interfere with the ligation by TasI DNA ligase. In the assay with G551D . CF, the l evel of misligation for the Normal target aequence was 5~ while that for R553X was 296. This illustratea the utility of introducing a mismatch at a site several bases away from the ligation junction in improving discrimination. Ultimately such a strategy may permit discrimination of mismatches not currently discr;m;n~te~l. This aspect may be illustrated by considering the assay with G551D. CF and T4 DNA
ligase. This enzyme did not discriminate the G-T
mismatch at 5' (1) . But the combination of mismatches that occurs in R553X was discr1m;n~t~d.

W095/27078 r~
2t 86465 TA;3LE VI
LIGATION SPI~CIFICITY IN THE G551D ASSAY
Probes Target Mismatch Per Cent Ligation 5 - T4l Taq~
.

G551D.CF NOR 5 ' (l) G-T 2796 5 G551D 309~ 4596 G551S 5' (1) G-T 1.096 0 5' (2) A-C
Q552X 5' (1) G-T 1.5~ 0.396 3 ' (2) T-G

R553X 5 ' (1) G-T 4% 2 3 ' (5) T-G
G551D.NOR NOR 43~ 539 G551D 5' (1) A-C 5.296 89 G551S 5' (2) A-C 5.4~ 4~
Q552X 3 ' (2) T-G 1. 996 296 R553X 3 ' (5) T-G 1096 5196 25 1600 mM NaCl ~lOO mM KCl/100 mM NaCl, 55C

Wo gs/27078 I ~ ~/~ ~

Example 11: Discrimination of Mismatches at Position8 Away from the I,igation JUnction A systematic evaluation of the abilities of T4 and Taq DNA ligases to discriminate mismatches at po8itions one base removed from the ligation junction was undertaken with the p53 model described in the patent application. These positions are r~ n~ted as 5' (2) and 3' (2) depending on the side of t~e ligation junction. The results are summarized in the enclosed figure. All mismatches were discriminated by both enzymes at the 3' (2) position. Not all 5' (2) mismatches were discriminated by either enzyme. (See Figure 18 . ) Example 12: Effect of Enzyme C~ nc~ntration on Ligation Specif icity Two different T4 DNA ligase concentrations were compared: 1 nM vs 240 nM. The ability of T4 DNA ligase to discriminate 5' (1) and 3' (1) mismatches in the p53 model was evaluated. The result9 are summarized in the ~igure. Clearly, the ligation specificity for 3' (1) -- misimatches improved at the lower enzyme concentration without significant 1088 of ligation for the complementary matches. There was also il.. ,)L~JV~ t for the 5' (1) mismatches, especially for the purine-purine ~1~ W095/27078 21~64~5 r~" e m; P'--t~ 'P., but not ag significant overall as for the 3 ' (1) mismatches. (See Figures 19 and 20. ) woss/27078 ?1 8646~ P~
SEQUENCE LISTlNG
(I) G~RAL INFORMATION
(i) APPLICANT: Ciba Corning Diagnostics Co Martinelli, Richard A. rp Arruda, John C.
(ii) TITLE OF I~VENTION: IIy~,iJ;~;~.~ Ligation As8ays for the Detection of Specific Nucleic Acid Sequences (iii) NUMBER OF SEQUENCES: 26 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRF,~!~F.F, Ciba-Corning Dagnostics Corp.
(13) STRFET: 63 North Street (C) CITY: Medfield (D) STATE r r , ,, (E) COUNTRY: USA
OZIP: 02052 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Diskette-3.~0 inch, 1.44Mb storage ~) IBM Compatible (C) OPERATING SYSTEM: IBM DOS 5.0 (D) SOFTWARE: WORD 6.0 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION N[JMBER: Not available (B) FILING DAT_: Not available (C) CLASSIFICATION: Not available W09~i/27078 r~ t r (vii) PRIOR APPLICATION DATA:
(A) APPLICATIONNUMBER: US 08/222,613 (13) FILING DATE: 04-04-1994 (C) CLASSIFICATION: Not available (vui) ATTORNEY/AGENT INFORMATION:
(A) NAME: I~ t~,..., A. S.
(B) REGISTRATION NUMBER: 28,244 (C) REFERENCEIDOCKET NU~D3ER: CCD-I 13 25 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 508-359-3836 ~3) TELEFAX: 508-359-3885 (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 47 bases (B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii)MOLECULETYPE GenomicDNA
(A) DESCRIPTION: Delta F508, a portion of the sequence of exon 10 of the CFTR gene b...l ~ base number 1652 withbase 1653-1655 deleted.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

wo9s/27078 2-1 86465 P ~

(3) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 bases (B)TYPE: Nucleicacid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: Genomic DNA
(A) DESCRIPTION: Normal, a portion of the sequence of exon 10 of the CFTR gene b~ ,, base number 1652 25 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

(4) INFORMATION FOR SEQ ID NO: 3:
C~) SEQUENCE CHARACTERISTICS
(A) LENGTH: 24 bases (13) TYPE: Nudeic acid (C) STRANDEDNESS: Smgle (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: Genomic DNA
(A) DESCRIPTION: C16B, bases 1611 - 1634 of exon 10 of the CFTR gene (xi) SEQUENCEDESCRIPTION: SEQ ~) NO: 3:

Wo ss/27078 2 1 ~646~ C

(5) INFORMATION FOR SEQ ID NO: 4:
~ 1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 bases (B) TYPE: Nucleic acid 15 (C) STRANDEDNESS: Single (D)TOPOLOGY: Linear (ii) MOLECULE TYPE: Genomic DNA
(A) DESCRIPTION: C16D, bases 1708 - 1684 of exon 10 of the CFTR gene (xi)SEQUENCEDESCRlPTlON: SEQIDNO: 4:

GTT G~C ATG CTT TGA TGA CGC TTC 24 (6) INFORMATION FOR SEQ ID NO: 5:
) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 53 bases (B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear i) MOLECULE TYPE: Other DNA/Genomic DNA
(A) DESCRIPTION: PMP.508, bases I - 29 is a spacer of synthetic DNA; bases 30 - 53 consists of bases 1 656-1678 of exon 10 of the CFIR gene (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

wo9~/27078 2 1 8 6465 (7) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARA(~ lC~:

(A) LENGTH: 24 bases (B) TYPE: Nucleic wid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: Genornic DNA
(A) DESCRIPTION: 508.CF, bases 1629-1652 of exon 10 of the CFTR gene (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
AT GAT ATT TTC m AAT GGT GCC A 24 (8) ~IFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 bases (B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear Cli) MOLECULE TYPE: Genomic DNA
(A) DESCRIPTION: 508.NOR, bases 1629-1655 of exon 10 of the CFTR gene (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

~0 (9) INFORMATION FOR SEQ ID NO: 8:

W095/27078 r~l,~c.~
21 ~6465 (i) SEQUENCE CEIARACTERISTICS:
(A) LENGl~l: 47 bases (13) TYPE: Nucleicacid (C) STRANDEDNESS: Sulgle (D) TOPOLOGY: Linear i) MOLECULE TYPE: Genomic DNA
(A) DESCRIPTION: Delta I-507, a portion of the sequence of exon 10 of the CFTR gene ~u..~ " ~ base number 1652 with base 1652-1654 deleted.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: ~:

(10) INFORMATION FOR SEQ ID NO: 9:
30 ~1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 56 bases (B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D)TOPOLOGY: Linear 40 ~u) MOLECULE TYPE: Genomic DNA / other DNA
(A) DESCRIPTION: PMP 507: bases I - 29 is a spacer of synthetic DNA; bases 30 - 56 consists of bases 1679 - 1653 of exon 10 of the CFTR gene (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

WO#127078 21364~5 P- ~ ~
s (I l) INFORMATION FOR SEQ ID NO: 10:
~I) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 bases (B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear ( i) MOLECULE TYPE: Genomic DNA
(A) DESCRIPTION: 507.CF, consists of bases 1626-1649 of exon 10 of the CFTR gene (xi) SEQUENCE DESCRIPTION: SEQ lD NO: 10:

(12) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 54 bases (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D)TOPOLOGY: Linear (ii) MOLECULE TYPE: Genomic DNA
(A) DESCRIPTION. G542X, bases 1731 - 1784 of exon 11 of thc CFTR gene with T substituted for G at base 1756 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

50 (13) INFORMATION FOR SEQ ID NO: 12:

~ Wossl27o78 2186465 r~

) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 54 bases (B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D)TOPOLOGY: Linear i) MOLECULE TYPE: Genornic DNA
(A) DESCRIPTION: NOR173 1.54, bases 1731 - 1784 of exon 11 of the CFTR gene (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

(14) INFORMATION FOR SEQ ID NO: 13:
~1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 52 bases (B) TYPE: Nucleic wid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear ~i) MOT .FCTTr .~. TYPE: Other DNA/Genornic DNA
(A) DESCRIPTION: PMP.G542X bases 1-27 consists of a spacer of synthetic DNA, bases 28 - 52 consists of bases 1781-1757 of exon 11 ofthe CFTR gene 45 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

wo 95~27078 P~

(15) INFORMATION FOR SEQ ID NO: 14:
) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 bases (B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear ( i) MOLECTJLE mE: Genon~ic DNA
(A) DESCR~PTION: G542X.CF, bases 1756 - 1730 of exon 11 of the C~TR gene with T substituted for C at base (xi) SEQUENCE DESCR~PTION: SEQ ID NO: 14:

(16) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 bases tl3) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D)TOPOLOGY: Linear ( i) MOT FcrlT F TYPE: Genomic DNA
(A) DESCRIPTION: G542X.NOR, bases 1756 - 1730 of exon 11 of the CFTR gene 45 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1~:

(17) INFORMATION FOR SEQ ID NO: 16:

21 ~6465 5 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 bases (13) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear 15 (ii) MOLECULE TYPE: Genomic DNA
(A) DESCRIPTION: G551D, bases 1763 - 1807 of on 11 of the CFTR gene with A substituted for the G at base (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

(18)1NFORMATIONFORSEQIDNO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 bases (13) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: Genomic DNA
(A) DESCRIPTION: NOR17632.45, bases 1 763 -1 807 0f on 11 of the CFTR gene (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

i5 (19) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:

Wo ss/2707s 2 1 8 6 ~ 6 5 (A) LENGT~: 45 bases (B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D)TOPOLOGY: Linear ~li) MOLECULE TYPE: Genomic DNA
(A) DESCR~TION: G551S, bases 1763 - 1807 of exon 11 of the CFTR gene with A substitued for G at base 1783 (xi) SEQUENOE DESCRIPTION: SEQ ID NO: 18:

(20) INFORMATION FOR SEQ ID NO: 19:
~1) SEQUENCE CHARACTERISTICS:
(A)LENGTH: 45bases (B) TYPE: Nudeic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear ~li) MOLECULE TYPE: Genomic DNA
(A) DESCRIPTION: Q552X bases 1763 - 1807 of exon 11 of the CFTR gene with T substitued for C at base 1786 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:

(21) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 52 bases (B) TYPE: Nucleic acid WO 95n7078 ~ 't (C) STRANDEDNESS: Single - (D)TOPOLOGY: Linear 10 (ii) MOLECULE TYPE: Other DNAlGenomic DNA
(A) DESCRIPTION: P~.G55 ID, bases 1-29 consist of a spacer of synthetic DNA, base6 30-52 consist of bases 1785-18070fexonll oftheCFTRgene (xi) SEQUENCE DESCRIPTION: SEQ ~ NO: 20:

(22) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 bases (B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (I)) TOPOLOGY: Linear ~li) MOLECULE TYPE: Genomic DNA
(A)DESCRIPTION: G551D.CF, bases 1784 -1763 of exon 11 of the CFTR gene with T substituted for C at base 40 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:

(23) INFORMATION FOR SEQ ID NO: 22:
) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 bases Q3)TYPE: Nucleicacid , w095/27078 21 ~6465 r~
(C) STRANDEDNESS: Single (l))TOPOLOGY: Linear 10 ~li) MOLECULE TYPE: Genomic DNA
(A)DESCRIPTION: G551DNOR, bases 1784-1763 of exon ll of the CFTR gene 15 (xi)SEQUENCEDESCRlPTlON: SEQIDNO: 22:

(24) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS:
(A)LENGTEI: 35bases (13) mE: Nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear i) MOLECULE TYPE: Genomic DNA
(A) DESCRIPTION: p53, 35 bases flanking codon 175 of the gene for p53 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:

40 (25) INFORMATION FOR SEQ ID NO: 24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 bases (B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D)TOPOLOGY: Linear W095/27078 r~l,., ~''~ ~

(ii) MOLECULE TYPE: Genomic DNA
(A) DESCRIPTION: pS3.5', The sequence of 16 bases extending 5' from the C of codon 1 75 of the pS3 gene.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:

15 (26) INFORMATION FOR SEQ ID NO: 25:
Cl) SEQUENCE CHARACTERISTICS:
(A)LENGTH: 19bases (B) mE: Nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (i;) MOT PCr~T.T~. TYPE: Genomic DNA
(A) L~ N: p53.3', The sequence of 19 bases extending 3' from the G of codon 175 of the p53 gene.
(xi) SEQTJENCE DESCRIPTION: SEQ ID NO: 25:

(27) INFORMATIONFORSEQIDNO: 26 ( ) SEQTJENCE CHARACTERISTICS
(A) LENGTH: 45 bases ~) TYPE: Nucleicacid (C) STANDEDNESS: Single Cli) MOT .T~.CT~T F TYPE: Genomic DNA
(A) DESCRlPTION: R553X bases 1763-1807 of exon 11 ofthe CFTR gene with T substituted for C at base 1789 W09s/27078 21 86465 P~ C ~
5 (ix) SEQUENt'F nF~f~RTpTIoN SEQ ID NO 26 (28) INFORMATIONFORSEQIDNO: 27 (i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 24 bases ~3) TYPE: Nucleicacid (C) STANDEDNESS: Single (ii) MOLECULE TYPE: Genomic DNA
(A) DESCRIPTION: CFI, bases 1656-1678 of exon 10 ofthe C-FTR
25 ~LlC) SEQUENCE DESCRIPTION SEQ ID NO 27

Claims

What is claimed is:
1. A method for identifying a target polynucleic acid sequence comprising:
(a) providing a target polynucleic acid, said target having a normal or mutated sequence;
(b) providing a first probe joined to a separating moiety and a second probe joined to a label, wherein if said first probe and said second probe are ligated said first probe and said second probe are completely complementary to said target if said target has a normal sequence, and wherein said first probe or said second probe have a mismatch with said target if said target has a mutated sequence;
(c) mixing said first probe and said second probe and said target so as to form a first reaction mixture, said mixing being done under conditions wherein said first probe and said second probe hybridize to said target even if a mismatch is present between said first probe or said second probe and said target;
(d) adding a ligating reagent to said first reaction mixture so as to form a second reaction mixture under conditions so as to join said first probe and said second probe if said first probe and said second probe match said target;
(e) separating said first probe from said second reaction mixture utilizing said separating moiety of said first probe; and (f) analyzing said separated first probe to determine if said label of said second probe is attached thereto.

2. A method of claim 1, further comprising a denaturing step after step (d) wherein said second reaction mixture is denatured so that said first probe and said second probe are separated from said target.
3. A method of claim 1 wherein the moiety that permits the probe to be separated is an insoluble particle.
4. A method of claim 1 wherein the moiety that permits the probe to be separated is a magnetic particle.
5. A method of claim 1 wherein the label is selected from the group consisting of enzymatic moieties, radioactive moieties, fluorescent moieties, luminescent moieties, and entities that permit subsequent attachment to a label.
6. A method of claim 5 wherein said entities that permit subsequent attachment to a label are avidin or biotin.
7. A method of claim 5 wherein the label is a luminescent material.
8. A method of claim 7 wherein the label is an acridinium ester.
9. A method of claim 1 in which the label is an acridinium ester and the analysis of the separated probe to determine the presence of the label comprises the addition of DNAase before addition of the flash reagent.

10. A method of claim 1 wherein the target polynucleic acid is selected from the group consisting of DNA or polymers thereof, RNA or polymers thereof, and viral material.
11. A method of claim 1 which also includes a step for amplifying the target polynucleic acid before it is mixed with said probes.
12. A method of claim 11 in which the amplification technique is selected from the group consisting of polymerase chain reaction, ligase chain reaction and QB replicase.
13. A method of claim 1 in which the ligating agent acts enzymatically or chemically to join the two probes.
14. A method of claim 13 in which the ligating agent is ligase.
15. A method of claim 1 in which sodium chloride is present during the ligating step, such sodium chloride being present at a concentration of 200 - 1000 mM.
16. A method of claim 15 in which the sodium chloride concentration is 500 - 700 mM.
17. A method of claim 16 in which the sodium chloride concentration is approximately 600 mM.
18. A method of claim 2 in which, after denaturation, the sample is passed through a chromatography column, said column then being analyzed to determine if the label is attached thereto.

23. A method of claim 1 wherein one of said probes is joined to a first subunit of the midivariant sequence, and said other probe is joined to a second subunit of the midivariant sequence, and wherein said separated first probe is analyzed to determine if said first probe has the ability to replicate if put in a reaction mixture containing QB replicase.
24. The method of claim 23 in which QB replicase is added along with the probes, and the analysis comprises determining if the probes had replicated.
25. A method of claim 1 further comprising providing at least one additional probe, wherein if said first probe and said second probe and said additional probe are ligated, said first probe, said second probe and said additional probe are completely complementary to said target if said target has a normal sequence, and wherein said first probe or said second probe or said additional probe have a mismatch with said target if said target has a mutated sequence.
26. A method of claim 1 in which the label is located in such a position that it does not interfere with hybridization and ligation.
27. A method of claim 2 which also includes the process wherein, before denaturation, an aliquot is removed, said aliquot being analyzed to determine if the label is hybridized to the target by a separating the probe containing the moiety that permits separation and the other entities attached thereto and b. analyzing said separated probe to determine if the label is attached thereto.
28. A method of claim 1 which also includes analyzing the supernatant remaining after separation of said probe containing said moiety that permits separation to analyze said supernatant for the presence of the label contained therein.
29. A method of claim 1 wherein the ligating reagent is selected from the group consisting of Taq DNA-ligase or T4 DNA-ligase.
30. The method of claim 29 in which the buffer containing said Taq DNA ligase also includes therein tRNA.
31. A method of claim 1 wherein the ligating reagent is added at the same time as the probes.
32. A method of claim 15 in which said sodium chloride is replaced by another salt or a mixture of salts.
33. A method of claim 1 wherein separating step (e) can take place either before or after ligating step (d).