CA2311333A1 - Methods for making nucleic acids - Google Patents
Methods for making nucleic acids Download PDFInfo
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- CA2311333A1 CA2311333A1 CA002311333A CA2311333A CA2311333A1 CA 2311333 A1 CA2311333 A1 CA 2311333A1 CA 002311333 A CA002311333 A CA 002311333A CA 2311333 A CA2311333 A CA 2311333A CA 2311333 A1 CA2311333 A1 CA 2311333A1
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- 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
- C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1096—Processes for the isolation, preparation or purification of DNA or RNA cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
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- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
Abstract
Nucleic acids are made by adding a known nucleotide sequence to the 3' end of a first RNA having a known sequence at the 5' end to form a second RNA and reverse transcribing the second RNA to form a cDNA. In one embodiment, the first RNA is an amplified mRNA, the known sequence at the 5' end comprises a poly(T) sequence, the adding step comprises using a polyadenyltransferase to add a poly(A) sequence to the 3' end, the reverse transcribing step is initiated at a duplex region comprising the poly(T) sequence hybridized to the poly(A) sequence, the cDNA is converted to double-stranded cDNA by a polymerase initiating from a noncovalently joined duplex region, and the double-stranded cDNA is transcribed to form one or more third RNAs.
Description
Methods for Making Nucleic Acids The disclosed inventions were made with Government support under Grant (Contract) Nos. GM07048, 1 RO1 DC02253 and SF32DC00193-03 awarded by the National Institutes of Health. The government may have rights in these inventions.
INTRODUCTION
Field of the Invention The field of this invention is making nucleic acids.
Background The ability to characterize cells by gene expression provides a wide variety of applications in therapy, diagnostics and biomedical technology. However, in many of these application, the starting or source material such as stem cells, cancerous cells, identified neurons, embryonic cells, etc. is highly limiting, making it necessary to amplify the targeted mRNA populations. Two existing methods for amplifying mRNA populations suffer from significant limitations. One method, the Brady and Iscove method (Brady et al., 1990, Methods Mol & Cell i3iol 2, I7-25), produces only short (200-300 bp), extreme 3' fragments of mRNAs using a PCR-based method which exponentially amplifies artifacts. A
second method, the Eberwine protocol (Eberwine et al. (1992) Proc.Natl.Acad.Sci USA
89, 3010-3014) provides sequential linear amplification steps and is the current method of choice for amplifying mRNA populations from limiting material. Nevertheless, this protocol suffers from a number of deficiencies. For example, the amplified product does not represent full-length aRNA for many endogenous mRNAs, and hence the method is of limited use for generating probes or cDNA libraries.
Relevant Literature Sippel (1973) Eur.J.Biochem. 37, 31-40 discloses the characterization of an ATP:RNA adenyltransferase from E. coli and Wittmann et al. (1997) Biochim.Biophys.Acta 1350, 293-305 disclose the characterization of a mammalian poly(A) polymerase.
Gething et al. (1980) Nature 287, 30I-306 disclose the use of an ATP:RNA
adenyltransferase to polyadenylate the '3 termini of total influenza virus RNA. Eberwine et al.
(1996) US Patent No.5,514,545 describes a method for characterizing single cells based on RNA
amplification. Eberwine et al. (1992) Proc.Natl.Acad.Sci USA 89, 3010-3014, describe the analysis of gene expression in single live neurons. Gubler U and Hoffman BJ.
(1983) Gene (2-3), 263-9, describe a method for generating cDNA libraries, see also the more recent reviews, Gubler (1987) Methods in Enzymology, 152, 325-329 and Gubler (1987) Methods in Enzymology, 152, 330-335. Clontech (Palo Alto, CA) produces a "Capfinder"
cloning kit that uses "GGG" primers against nascent cDNAs capped with by reverse transcriptase, Clontechniques 11, 2-3 (Oct 1996), see also Maleszka et al. (1997) Gene 202, 39-43.
SUMMARY OF THE INVENTION
The invention provides methods and compositions for making nucleic acids. The general methods comprise the steps of adding a known nucleotide sequence to the 3' end of a first RNA having a known sequence at the 5' end to form a second RNA and reverse transcribing the second RNA to form a cDNA. According to one embodiment, the first RNA
is an amplified mRNA, the known sequence at the 5' end comprises a poly(T) sequence, the adding step comprises using a polyadenyltransferase to add a poly(A) sequence to the 3' end, and the reverse transcribing step is initiated at a duplex region comprising the poly(T}
sequence hybridized to the poly(A) sequence. The resultant cDNA transcript may be single-stranded, isolated from the second RNA and optionally converted to double-stranded cDNA, preferably by a DNA polymerase initiating at a noncovalently joined duplex region. The cDNA may also be transcribed to form one or more third RNAs. In another embodiment, the first RNA is made by amplifying a mRNA by the steps of hybridizing to the poly(A) tail of the mRNA a poly{T) oligonucleotide joined to an RNA polymerase promoter sequence, reverse transcribing the mRNA to form single-stranded cDNA, converting the single-stranded cDNA to a double-stranded cDNA and transcribing the double-stranded cDNA to form the first RNA.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 is a schematic of one embodiment of the invention for amplifying mRNA.
Figure 2 is a schematic of another embodiment of the invention using a second promoter sequence.
INTRODUCTION
Field of the Invention The field of this invention is making nucleic acids.
Background The ability to characterize cells by gene expression provides a wide variety of applications in therapy, diagnostics and biomedical technology. However, in many of these application, the starting or source material such as stem cells, cancerous cells, identified neurons, embryonic cells, etc. is highly limiting, making it necessary to amplify the targeted mRNA populations. Two existing methods for amplifying mRNA populations suffer from significant limitations. One method, the Brady and Iscove method (Brady et al., 1990, Methods Mol & Cell i3iol 2, I7-25), produces only short (200-300 bp), extreme 3' fragments of mRNAs using a PCR-based method which exponentially amplifies artifacts. A
second method, the Eberwine protocol (Eberwine et al. (1992) Proc.Natl.Acad.Sci USA
89, 3010-3014) provides sequential linear amplification steps and is the current method of choice for amplifying mRNA populations from limiting material. Nevertheless, this protocol suffers from a number of deficiencies. For example, the amplified product does not represent full-length aRNA for many endogenous mRNAs, and hence the method is of limited use for generating probes or cDNA libraries.
Relevant Literature Sippel (1973) Eur.J.Biochem. 37, 31-40 discloses the characterization of an ATP:RNA adenyltransferase from E. coli and Wittmann et al. (1997) Biochim.Biophys.Acta 1350, 293-305 disclose the characterization of a mammalian poly(A) polymerase.
Gething et al. (1980) Nature 287, 30I-306 disclose the use of an ATP:RNA
adenyltransferase to polyadenylate the '3 termini of total influenza virus RNA. Eberwine et al.
(1996) US Patent No.5,514,545 describes a method for characterizing single cells based on RNA
amplification. Eberwine et al. (1992) Proc.Natl.Acad.Sci USA 89, 3010-3014, describe the analysis of gene expression in single live neurons. Gubler U and Hoffman BJ.
(1983) Gene (2-3), 263-9, describe a method for generating cDNA libraries, see also the more recent reviews, Gubler (1987) Methods in Enzymology, 152, 325-329 and Gubler (1987) Methods in Enzymology, 152, 330-335. Clontech (Palo Alto, CA) produces a "Capfinder"
cloning kit that uses "GGG" primers against nascent cDNAs capped with by reverse transcriptase, Clontechniques 11, 2-3 (Oct 1996), see also Maleszka et al. (1997) Gene 202, 39-43.
SUMMARY OF THE INVENTION
The invention provides methods and compositions for making nucleic acids. The general methods comprise the steps of adding a known nucleotide sequence to the 3' end of a first RNA having a known sequence at the 5' end to form a second RNA and reverse transcribing the second RNA to form a cDNA. According to one embodiment, the first RNA
is an amplified mRNA, the known sequence at the 5' end comprises a poly(T) sequence, the adding step comprises using a polyadenyltransferase to add a poly(A) sequence to the 3' end, and the reverse transcribing step is initiated at a duplex region comprising the poly(T}
sequence hybridized to the poly(A) sequence. The resultant cDNA transcript may be single-stranded, isolated from the second RNA and optionally converted to double-stranded cDNA, preferably by a DNA polymerase initiating at a noncovalently joined duplex region. The cDNA may also be transcribed to form one or more third RNAs. In another embodiment, the first RNA is made by amplifying a mRNA by the steps of hybridizing to the poly(A) tail of the mRNA a poly{T) oligonucleotide joined to an RNA polymerase promoter sequence, reverse transcribing the mRNA to form single-stranded cDNA, converting the single-stranded cDNA to a double-stranded cDNA and transcribing the double-stranded cDNA to form the first RNA.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 is a schematic of one embodiment of the invention for amplifying mRNA.
Figure 2 is a schematic of another embodiment of the invention using a second promoter sequence.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION
The following preferred embodiments and examples are offered by way of illustration and not by way of limitation.
The general methods comprise the steps of adding a known nucleotide sequence to the 3' end of a first RNA having a known sequence at the 5' end to form a second RNA and reverse transcribing the second RNA to form a cDNA. The known sequence at the 5' end of the first RNA species is sufficient to provide a target for a primer and otherwise determined largely by the nature of the starting material. For example, where the starting material is mRNA, the known sequence at the 5' end may comprise a poly(A) sequence and/or (b) an internal mRNA sequence of an mRNA. Alternatively, where the starting material is amplified RNA, or aRNA, the known sequence may comprise a poly(T) sequence or the complement of a known internal mRNA sequence. The known 5' sequence may advantageously comprise additional sequences such as primer target sites, RNA
polymerase sites, etc. For example, the presence of both a primer target site such as a poly(T) sequence and an RNA polymerase promoter sequence permits enhanced opportunities for downstream amplification or transcription (see Figure 2 and related text below).
The adding step may be effect by any convenient method. For example, a polyadenyltransferase or poly(A) polymerase may be used to add selected nucleotides to the 3' end. Poly(A) polymerases may be derived from a wide variety of prokaryotic and eukaryotic sources, are commercially available and well-characterized. In another example, a ligase may be used to add one or more selected oligonucleotides. These enzymes are similarly readily and widely available from a wide variety of sources and are well characterized.
The added known 3' sequence is similarly sufficient to provide a target for a primer, otherwise the nature of the added known sequence is a matter of convenience, limited only by the addition method. For example, using ligase mediated oligonucleotide addition, essentially any known sequence that can be used as target for a primer may be added to the 3' end. With polyadenyltransferase mediated addition, it is generally more convenient to add a poly(N) sequence, with many such transferases demonstrating optimal efficiency when adding poly(A) sequence. Fore poiyadenyltransferase mediated additions, the added sequence will generally be in the range of 5 to 50 nucleotides, preferably in the range of 6 to 25 nucleotides, more preferably in the range of 7 to 15 nucleotides.
The reverse transcribing step is initiated at a noncovalently joined duplex region at or near the '3 end of the second RNA species (the first species with the added 3' sequence), generally formed by adding a primer having sufficient complementarity to the 3' end sequence to hybridize thereto. Hence, where the 3' end comprises a poly(A) sequence, the reverse transcribing step is preferably initiated at a duplex region comprising a poly(T) sequence hybridized to the poly(A) sequence. For many applications, the primer comprises additional functional sequence such as one or more RNA polymerise promoter sequences such as a T7 or T3 RNA polymerise promoter, one or more primer sequences, etc.
In a preferred embodiment, the RNA polymerise promoter sequence is a T7 RNA
polymerise promoter sequence comprising at least nucleotides -17 to +6 of a wild-type T7 RNA polymerise promoter sequence, preferably joined to at least 20, preferably at least 30 nucleotides of upstream flanking sequence, particularly upstream T7 RNA
polymerise promoter flanking sequence. Additional downstream flanking sequence, particularly downstream T7 RNA polymerise promoter flanking sequence, e.g. nucleotides +7 to +10, may also be advantageously used. For example, in one particular embodiment, the promoter comprises nucleotides -50 to +10 of a natural class III T7 RNA polymerise promoter sequence. Table 1 prcivides exemplary promoter sequences and their relative transcriptional efficiencies in the subject methods (the recited promoter sequences are joined to a 23 nucleotide natural class III T7 promoter upstream flanking sequence).
Table I. Transcriptional efficiency of T7 RNA polymerise promoter sequences.
Promoter Sequence Transcriptional Efficiency T AAT ACG ACT CAC TAT AGG GAG A ++++
(SEQ ID NO:1, class III T7 RNA polymerise promoter) T AAT ACG ACT CAC TAT AGG CGC +
(SEQ ID N0:2, Eberwine et al. ( 1992) supra) T AAT ACG ACT CAC TAT AGG GCG A +
(SEQ ID N0:3, Bluescript, Stratagene, La Jolla, CA) The transcribed cDNA is initially single-stranded and may be isolated from the second RNA by any of wide variety of established methods. For example, the method may involve treating the RNA with a nuclease such as RNase H, a denaturant such as heat or an alkali, etc., and/or separating the strands electrophoretically. The second strand cDNA
synthesis may be effected by a number of well established techniques including 3'-terminal hairpin loop priming or methods wherein the polymerization is initiated at a noncovalently joined duplex region, generated for example, by adding exogenous primer complementary to the 3' end of the first cDNA strand or in the course of the Hoffman-Gubler protocol. In this latter embodiment, the cDNA isolation and conversion to double-stranded cDNA
steps may be effected together, e.g: contacting the RNA with an RNase H and contacting the single-stranded cDNA with a DNA polymerase in a single incubation step. In any event, these methods can be used to construct cDNA libraries from very small, e.g. single cell, starting materials.
In a particular embodiment, the methods further comprise the step of repeatedly transcribing the single or double-stranded cDNA to form a plurality of third RNAs, in effect, amplifying the first RNA species. Preferred transcription conditions employ a class III T7 promoter sequence (SEQ ID NO:1 ) and a T7 RNA polymerase under the following reaction conditions: 40mM Tris pH 7.9, 6mM MgCl2, 2mM Spermidine, IOmM DTT, 2mM NTP
(Pharmacia), 40 units RNAsin (Promega), 300-1000 units T7 RNA Polymerase (6.16 Prep).
The enzyme is stored in 20 mM HEPES pH 7.5, 100 mM NaCI, 1 mM EDTA, 1 mM DTT
and 50% Glycerol at a protein concentration of 2.5 mg/mL and an activity of units/uL. In exemplary demonstrations, 1-3 uL of this polymerase was used in 50 uL
reactions. Starting concentrations of template can vary from picogram quantities (single cell level) to 1 ug or more of linear plasmid DNA. The final NaCI concentration is preferably not higher than 6 mM.
In a more particular embodiment, the first RNA is itself made by amplifying an RNA, preferably a mRNA. For example, the first RNA may be made by amplifying a mRNA by the steps of hybridizing to the poly(A) tail of the mRNA a poly(T) oligonucleotide joined to an RNA polymerase promoter sequence, reverse transcribing the mRNA to form single-stranded cDNA, converting the single-stranded cDNA to a double-stranded cDNA and transcribing the double-stranded cDNA to form the first RNA.
Figure 1 is a schematic of this serial mRNA amplification embodiment of the invention, highlighting individual steps of the method:
(a) An oligonucleotide primer, consisting of 5'-T~-RNA polymerase promoter-oligo (dT)24-3', is annealed to the poiy(A) tract present at the 3' end of mature mRNAs, and first-strand cDNA is synthesized using reverse transcriptase, yielding an RNA-DNA
hybrid (RNA
is denoted by open boxes; DNA by filled boxes);
(b) The hybrid is treated with RNase H, DNA polymerise, and DNA ligase to convert the single-stranded cDNA into double-stranded cDNA;
(c) T, RNA polymerise is used to synthesize large amounts of amplified RNA
(aRNA) from this cDNA. The incorporation of a modified T~ polymerise promoter sequence into our primer, as compared to the altered promoter sequence utilized by Eberwine et al., PNAS 89: 3010-3014, 1992, greatly increases the yield of aRNA;
(d) The aRNA is tailed with poly(A) using a poly(A) polymerise. This modification generates much longer first-strand cDNA in the next step as compared to the original protocol;
(e) After denaturation and elimination of the aRNA, a T; RNA polymerise promoter-oligo (dT) primer is annealed to this newly synthesized poly(A) sequence, and reverse transcriptase is used to synthesize first-strand cDNA. Second-strand cDNA and the complementary strand of the polymerise promoter are synthesized as in (b); and (f) T, RNA polymerise is then used to generate aRNA from this cDNA template.
Another embodiment involves the incorporation of additional sequences during certain synthesis steps. These sequences allow, for example, for the PCR
amplification of the amplified RNA, for direct second-round amplification without synthesizing a full second strand cDNA, etc. This embodiment is diagramed in Figure 2:
(a) This is step (a) of Figure 1, except that the primer for first strand cDNA
synthesis also includes a promoter site for a different RNA polymerise (shown with SP6;
polymerise site is also possible) between the poly(T) and the T~ sequences;
(b) This is step (b) of Figure 1;
(c) This is step (c) of Figure l, except that the aRNA now has an RNA
polymerise site at its 5' end;
(d) This is step (d) of Figure 1;
(e) This is step (e) of Figure 1, except that the oligonucleotide used for priming first strand cDNA synthesis also has an additional sequence at its S' end suitable for use as a priming site during polymerise chain reaction (PCR). Note also that the SP6 or polymerise site has been copied into first strand cDNA. Because this first strand cDNA has unique sequences at both its 5' and 3' ends, it can now be used directly in a PCR reaction for total amplification of all sequences, as an alternative to performing another round of aRNA
synthesis;
(f) The first strand cDNA can be used directly for aRNA synthesis by annealing an oligonucleotide incorporating the complementary portion of the SP6 or preferably, the T3 RNA polymerase site. Or, the first strand cDNA can be converted into double-stranded cDNA through second strand synthesis, with aRNA synthesis then following.
All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Serafini, Tito Luu, Percy Lin, David S Ngai, John (ii) TITLE OF INVENTION: Methods for Making Nucleic Acids (iii) NUMBER OF SEQUENCES: 3 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: SCIENCE & TECHNOLOGY LAW GROUP
IO (B) STREET: 75 DENISE DRIVE
(C) CITY: HILLSBOROUGH
(D) STATE: CALIFORNIA
(E) COUNTRY: USA
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(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 ZO (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEYjAGENT INFORMATION:
25 (A) NAME: OSMAN, RICHARD A
(B) REGISTRATION NUMBER: 36,627 (C) REFERENCE/DOCKET NUMBER: B98-009 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (650) 343-4341 3O (B) TELEFAX: (650) 343-4342 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs 3S (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:
S (2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
IS (2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single 2~ (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
The following preferred embodiments and examples are offered by way of illustration and not by way of limitation.
The general methods comprise the steps of adding a known nucleotide sequence to the 3' end of a first RNA having a known sequence at the 5' end to form a second RNA and reverse transcribing the second RNA to form a cDNA. The known sequence at the 5' end of the first RNA species is sufficient to provide a target for a primer and otherwise determined largely by the nature of the starting material. For example, where the starting material is mRNA, the known sequence at the 5' end may comprise a poly(A) sequence and/or (b) an internal mRNA sequence of an mRNA. Alternatively, where the starting material is amplified RNA, or aRNA, the known sequence may comprise a poly(T) sequence or the complement of a known internal mRNA sequence. The known 5' sequence may advantageously comprise additional sequences such as primer target sites, RNA
polymerase sites, etc. For example, the presence of both a primer target site such as a poly(T) sequence and an RNA polymerase promoter sequence permits enhanced opportunities for downstream amplification or transcription (see Figure 2 and related text below).
The adding step may be effect by any convenient method. For example, a polyadenyltransferase or poly(A) polymerase may be used to add selected nucleotides to the 3' end. Poly(A) polymerases may be derived from a wide variety of prokaryotic and eukaryotic sources, are commercially available and well-characterized. In another example, a ligase may be used to add one or more selected oligonucleotides. These enzymes are similarly readily and widely available from a wide variety of sources and are well characterized.
The added known 3' sequence is similarly sufficient to provide a target for a primer, otherwise the nature of the added known sequence is a matter of convenience, limited only by the addition method. For example, using ligase mediated oligonucleotide addition, essentially any known sequence that can be used as target for a primer may be added to the 3' end. With polyadenyltransferase mediated addition, it is generally more convenient to add a poly(N) sequence, with many such transferases demonstrating optimal efficiency when adding poly(A) sequence. Fore poiyadenyltransferase mediated additions, the added sequence will generally be in the range of 5 to 50 nucleotides, preferably in the range of 6 to 25 nucleotides, more preferably in the range of 7 to 15 nucleotides.
The reverse transcribing step is initiated at a noncovalently joined duplex region at or near the '3 end of the second RNA species (the first species with the added 3' sequence), generally formed by adding a primer having sufficient complementarity to the 3' end sequence to hybridize thereto. Hence, where the 3' end comprises a poly(A) sequence, the reverse transcribing step is preferably initiated at a duplex region comprising a poly(T) sequence hybridized to the poly(A) sequence. For many applications, the primer comprises additional functional sequence such as one or more RNA polymerise promoter sequences such as a T7 or T3 RNA polymerise promoter, one or more primer sequences, etc.
In a preferred embodiment, the RNA polymerise promoter sequence is a T7 RNA
polymerise promoter sequence comprising at least nucleotides -17 to +6 of a wild-type T7 RNA polymerise promoter sequence, preferably joined to at least 20, preferably at least 30 nucleotides of upstream flanking sequence, particularly upstream T7 RNA
polymerise promoter flanking sequence. Additional downstream flanking sequence, particularly downstream T7 RNA polymerise promoter flanking sequence, e.g. nucleotides +7 to +10, may also be advantageously used. For example, in one particular embodiment, the promoter comprises nucleotides -50 to +10 of a natural class III T7 RNA polymerise promoter sequence. Table 1 prcivides exemplary promoter sequences and their relative transcriptional efficiencies in the subject methods (the recited promoter sequences are joined to a 23 nucleotide natural class III T7 promoter upstream flanking sequence).
Table I. Transcriptional efficiency of T7 RNA polymerise promoter sequences.
Promoter Sequence Transcriptional Efficiency T AAT ACG ACT CAC TAT AGG GAG A ++++
(SEQ ID NO:1, class III T7 RNA polymerise promoter) T AAT ACG ACT CAC TAT AGG CGC +
(SEQ ID N0:2, Eberwine et al. ( 1992) supra) T AAT ACG ACT CAC TAT AGG GCG A +
(SEQ ID N0:3, Bluescript, Stratagene, La Jolla, CA) The transcribed cDNA is initially single-stranded and may be isolated from the second RNA by any of wide variety of established methods. For example, the method may involve treating the RNA with a nuclease such as RNase H, a denaturant such as heat or an alkali, etc., and/or separating the strands electrophoretically. The second strand cDNA
synthesis may be effected by a number of well established techniques including 3'-terminal hairpin loop priming or methods wherein the polymerization is initiated at a noncovalently joined duplex region, generated for example, by adding exogenous primer complementary to the 3' end of the first cDNA strand or in the course of the Hoffman-Gubler protocol. In this latter embodiment, the cDNA isolation and conversion to double-stranded cDNA
steps may be effected together, e.g: contacting the RNA with an RNase H and contacting the single-stranded cDNA with a DNA polymerase in a single incubation step. In any event, these methods can be used to construct cDNA libraries from very small, e.g. single cell, starting materials.
In a particular embodiment, the methods further comprise the step of repeatedly transcribing the single or double-stranded cDNA to form a plurality of third RNAs, in effect, amplifying the first RNA species. Preferred transcription conditions employ a class III T7 promoter sequence (SEQ ID NO:1 ) and a T7 RNA polymerase under the following reaction conditions: 40mM Tris pH 7.9, 6mM MgCl2, 2mM Spermidine, IOmM DTT, 2mM NTP
(Pharmacia), 40 units RNAsin (Promega), 300-1000 units T7 RNA Polymerase (6.16 Prep).
The enzyme is stored in 20 mM HEPES pH 7.5, 100 mM NaCI, 1 mM EDTA, 1 mM DTT
and 50% Glycerol at a protein concentration of 2.5 mg/mL and an activity of units/uL. In exemplary demonstrations, 1-3 uL of this polymerase was used in 50 uL
reactions. Starting concentrations of template can vary from picogram quantities (single cell level) to 1 ug or more of linear plasmid DNA. The final NaCI concentration is preferably not higher than 6 mM.
In a more particular embodiment, the first RNA is itself made by amplifying an RNA, preferably a mRNA. For example, the first RNA may be made by amplifying a mRNA by the steps of hybridizing to the poly(A) tail of the mRNA a poly(T) oligonucleotide joined to an RNA polymerase promoter sequence, reverse transcribing the mRNA to form single-stranded cDNA, converting the single-stranded cDNA to a double-stranded cDNA and transcribing the double-stranded cDNA to form the first RNA.
Figure 1 is a schematic of this serial mRNA amplification embodiment of the invention, highlighting individual steps of the method:
(a) An oligonucleotide primer, consisting of 5'-T~-RNA polymerase promoter-oligo (dT)24-3', is annealed to the poiy(A) tract present at the 3' end of mature mRNAs, and first-strand cDNA is synthesized using reverse transcriptase, yielding an RNA-DNA
hybrid (RNA
is denoted by open boxes; DNA by filled boxes);
(b) The hybrid is treated with RNase H, DNA polymerise, and DNA ligase to convert the single-stranded cDNA into double-stranded cDNA;
(c) T, RNA polymerise is used to synthesize large amounts of amplified RNA
(aRNA) from this cDNA. The incorporation of a modified T~ polymerise promoter sequence into our primer, as compared to the altered promoter sequence utilized by Eberwine et al., PNAS 89: 3010-3014, 1992, greatly increases the yield of aRNA;
(d) The aRNA is tailed with poly(A) using a poly(A) polymerise. This modification generates much longer first-strand cDNA in the next step as compared to the original protocol;
(e) After denaturation and elimination of the aRNA, a T; RNA polymerise promoter-oligo (dT) primer is annealed to this newly synthesized poly(A) sequence, and reverse transcriptase is used to synthesize first-strand cDNA. Second-strand cDNA and the complementary strand of the polymerise promoter are synthesized as in (b); and (f) T, RNA polymerise is then used to generate aRNA from this cDNA template.
Another embodiment involves the incorporation of additional sequences during certain synthesis steps. These sequences allow, for example, for the PCR
amplification of the amplified RNA, for direct second-round amplification without synthesizing a full second strand cDNA, etc. This embodiment is diagramed in Figure 2:
(a) This is step (a) of Figure 1, except that the primer for first strand cDNA
synthesis also includes a promoter site for a different RNA polymerise (shown with SP6;
polymerise site is also possible) between the poly(T) and the T~ sequences;
(b) This is step (b) of Figure 1;
(c) This is step (c) of Figure l, except that the aRNA now has an RNA
polymerise site at its 5' end;
(d) This is step (d) of Figure 1;
(e) This is step (e) of Figure 1, except that the oligonucleotide used for priming first strand cDNA synthesis also has an additional sequence at its S' end suitable for use as a priming site during polymerise chain reaction (PCR). Note also that the SP6 or polymerise site has been copied into first strand cDNA. Because this first strand cDNA has unique sequences at both its 5' and 3' ends, it can now be used directly in a PCR reaction for total amplification of all sequences, as an alternative to performing another round of aRNA
synthesis;
(f) The first strand cDNA can be used directly for aRNA synthesis by annealing an oligonucleotide incorporating the complementary portion of the SP6 or preferably, the T3 RNA polymerase site. Or, the first strand cDNA can be converted into double-stranded cDNA through second strand synthesis, with aRNA synthesis then following.
All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Serafini, Tito Luu, Percy Lin, David S Ngai, John (ii) TITLE OF INVENTION: Methods for Making Nucleic Acids (iii) NUMBER OF SEQUENCES: 3 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: SCIENCE & TECHNOLOGY LAW GROUP
IO (B) STREET: 75 DENISE DRIVE
(C) CITY: HILLSBOROUGH
(D) STATE: CALIFORNIA
(E) COUNTRY: USA
(F) ZIP: 94010 IS (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 ZO (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEYjAGENT INFORMATION:
25 (A) NAME: OSMAN, RICHARD A
(B) REGISTRATION NUMBER: 36,627 (C) REFERENCE/DOCKET NUMBER: B98-009 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (650) 343-4341 3O (B) TELEFAX: (650) 343-4342 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs 3S (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:
S (2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
IS (2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single 2~ (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
Claims (21)
1. A method for making a nucleic acid comprising the steps of adding a known nucleotide sequence to the 3' end of a first RNA having a known sequence at the 5' end to form a second RNA and reverse transcribing the second RNA to form a cDNA.
2. A method according to claim 1, wherein the adding step comprises contacting the first RNA with at least one of (a) a nucleotide and polyadenyltransferase and (b) an oligonucleotide and a lipase.
3. A method according to claim 1, wherein the known sequence at the 3' end comprises a poly(A) sequence.
4. A method according to claim 1, wherein the known sequence at the 5' end comprises at least one of (a) a poly(T) or poly(A) sequence and (b) an internal sequence of an mRNA or the complement thereof.
5. A method according to claim 1, wherein the known sequence at the 5' end comprises a poly(T) sequence and an RNA polymerase promoter sequence.
6. A method according to claim 1, wherein the known sequence at the 3' end comprises a poly(A) sequence and the reverse transcribing step is initiated at a noncovalently joined duplex region comprising a poly(T) sequence hybridized to the poly(A) sequence.
7. A method according to claim 1, wherein the known sequence at the 3' end comprises a poly(A) sequence and the reverse transcribing step is initiated at a noncovalently joined duplex region comprising a poly(T) sequence hybridized to the poly(A) sequence, wherein the poly(T) sequence is covalently joined to at least one of a RNA polymerase promoter sequence and a primer sequence.
8. A method according to claim 1, wherein the cDNA is single-stranded and isolated from the second RNA.
9. A method according to claim 1, wherein the cDNA is single-stranded and isolated from the second RNA by a method comprising the step of contacting the RNA with at least one of an RNase H, a denaturant, and an alkali.
10. A method according to claim 1, wherein the cDNA is single-stranded and converted to a double-stranded cDNA.
11. A method according to claim 1, wherein the cDNA is single-stranded and converted to a double-stranded cDNA and the conversion is initiated at a noncovalently joined duplex region.
12. A method according to claim 1, wherein the cDNA is single-stranded and converted to a double-stranded cDNA by a method comprising the steps of contacting the RNA with an RNase H and contacting the single-stranded cDNA with a DNA polymerase whereby the DNA polymerase initiates the conversion at a noncovalently joined duplex region.
13. A method according to claim 1, wherein the cDNA is single-stranded and converted to a double-stranded cDNA by a method comprising the steps of contacting the RNA with a denaturant and contacting the single-stranded cDNA with a DNA polymerase and an oligonucleotide primer comprising a sequence complementary to the 3' end of the single-stranded cDNA, whereby the DNA polymerase initiates the conversion at a noncovalently joined duplex region of the 3' end of the single-stranded cDNA and the oligonucleotide primer.
14. A method according to claim 1, wherein the cDNA is single-stranded and converted to a double-stranded cDNA by a method comprising the steps of contacting the RNA with a denaturant and contacting the single-stranded cDNA with a DNA polymerase and an oligonucleotide primer comprising a sequence complementary to the 3' end of the single-stranded cDNA and an RNA polymerase promoter, whereby the DNA
polymerase initiates the conversion at a-noncovalently joined duplex region of the 3' end of the single-stranded cDNA and the oligonucleotide primer.
polymerase initiates the conversion at a-noncovalently joined duplex region of the 3' end of the single-stranded cDNA and the oligonucleotide primer.
15. A method according to claim 1, wherein the cDNA is single-stranded and converted to a double-stranded cDNA by a method comprising the steps of contacting the RNA with a denaturant and contacting the single-stranded cDNA with a DNA polymerase and an oligonucleotide primer comprising a sequence complementary to the 3' end of the single-stranded cDNA and an RNA polymerase promoter comprising a natural class polymerase promoter sequence, whereby the DNA polymerase initiates the conversion at a noncovalently joined duplex region of the 3' end of the single-stranded cDNA
and the oligonucleotide primer.
and the oligonucleotide primer.
16. A method according to claim 1, wherein the cDNA is single-stranded and converted to a double-stranded cDNA by a method comprising the steps of contacting the RNA with a denaturant and contacting the single-stranded cDNA with a DNA polymerase and an oligonucleotide primer comprising a sequence complementary to the 3' end of the single-stranded cDNA and an RNA polymerase promoter comprising SEQ ID NO:1 joined to an upstream flanking sequence of about 3 to 100 nucleotides. whereby the DNA
polymerase initiates the conversion at a noncovalently joined duplex region of the 3' end of the single-stranded cDNA and the oligonucleotide primer.
polymerase initiates the conversion at a noncovalently joined duplex region of the 3' end of the single-stranded cDNA and the oligonucleotide primer.
17. A method according to claim 1. further comprising the step of repeatedly transcribing the cDNA to form a plurality of third RNAs.
18. A method according to claim 1. wherein the cDNA is single-stranded and converted to a double-stranded cDNA, and the method further comprises the step of repeatedly transcribing the double-stranded cDNA to form a plurality of third RNAs.
19. A method according to claim 1, wherein the first RNA is made by amplifying a mRNA.
20. A method according to claim 1, wherein the first RNA is made by amplifying a mRNA by the steps of hybridizing to the poly(A)-tail of the mRNA a poly(T) oligonucleotide joined to an RNA polymerase promoter sequence, reverse transcribing the mRNA to form single-stranded cDNA, converting the single-stranded cDNA to a double-stranded cDNA and transcribing the double-stranded cDNA to form the first RNA.
21. A method according to claim 1, wherein the adding step comprises contacting the first RNA with a nucleotide and polyadenyltransferase.
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US6958997P | 1997-12-12 | 1997-12-12 | |
| US60/069,589 | 1997-12-12 | ||
| US09/049,806 US6114152A (en) | 1997-12-12 | 1998-03-27 | Methods for making nucleic acids |
| US09/049,806 | 1998-03-27 | ||
| PCT/US1998/026806 WO1999029907A1 (en) | 1997-12-12 | 1998-12-14 | Methods for making nucleic acids |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2311333A1 true CA2311333A1 (en) | 1999-06-17 |
Family
ID=26727557
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002311333A Abandoned CA2311333A1 (en) | 1997-12-12 | 1998-12-14 | Methods for making nucleic acids |
Country Status (6)
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| US (1) | US6114152A (en) |
| EP (1) | EP1038032A1 (en) |
| JP (1) | JP2001526053A (en) |
| AU (1) | AU733092B2 (en) |
| CA (1) | CA2311333A1 (en) |
| WO (1) | WO1999029907A1 (en) |
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| EP1056884B9 (en) * | 1998-02-27 | 2002-07-24 | PamGene B.V. | Method for the non-specific amplification of nucleic acid |
| US6794138B1 (en) * | 1999-12-16 | 2004-09-21 | Affymetrix, Inc. | Methods of small sample amplification |
| US6982143B1 (en) * | 2000-06-20 | 2006-01-03 | The Regents Of The University Of California | Normalizing and amplifying RNA |
| US20040161741A1 (en) * | 2001-06-30 | 2004-08-19 | Elazar Rabani | Novel compositions and processes for analyte detection, quantification and amplification |
| US9777312B2 (en) | 2001-06-30 | 2017-10-03 | Enzo Life Sciences, Inc. | Dual polarity analysis of nucleic acids |
| US6872529B2 (en) * | 2001-07-25 | 2005-03-29 | Affymetrix, Inc. | Complexity management of genomic DNA |
| US7297778B2 (en) | 2001-07-25 | 2007-11-20 | Affymetrix, Inc. | Complexity management of genomic DNA |
| US20060009409A1 (en) | 2002-02-01 | 2006-01-12 | Woolf Tod M | Double-stranded oligonucleotides |
| EP3415625A1 (en) | 2002-02-01 | 2018-12-19 | Life Technologies Corporation | Double-stranded oligonucleotides |
| WO2003064621A2 (en) | 2002-02-01 | 2003-08-07 | Ambion, Inc. | HIGH POTENCY siRNAS FOR REDUCING THE EXPRESSION OF TARGET GENES |
| AU2003276666A1 (en) * | 2002-06-12 | 2003-12-31 | Ambion, Inc. | Methods and compositions relating to polypeptides with rnase iii domains that mediate rna interference |
| US20040248094A1 (en) * | 2002-06-12 | 2004-12-09 | Ford Lance P. | Methods and compositions relating to labeled RNA molecules that reduce gene expression |
| CA2501983A1 (en) * | 2002-10-30 | 2004-05-27 | Pamgene B.V. | Improved methods for generating multiple rna copies |
| WO2005064019A2 (en) * | 2003-12-23 | 2005-07-14 | Genomic Health, Inc. | Universal amplification of fragmented rna |
| US7575863B2 (en) * | 2004-05-28 | 2009-08-18 | Applied Biosystems, Llc | Methods, compositions, and kits comprising linker probes for quantifying polynucleotides |
| DE602005025782D1 (en) * | 2004-09-21 | 2011-02-17 | Life Technologies Corp | TWO-TONE REAL-TIME / END POINT QUANTIFICATION OF MICRO-RNAS (MIRNAS) |
| US20060142228A1 (en) * | 2004-12-23 | 2006-06-29 | Ambion, Inc. | Methods and compositions concerning siRNA's as mediators of RNA interference |
| WO2006086668A2 (en) * | 2005-02-09 | 2006-08-17 | Epicentre Technologies | Compositions and methods employing 5'-phosphate-dependent nucleic acid exonucleases |
| US20080038727A1 (en) * | 2006-03-10 | 2008-02-14 | Applera Corporation | MicroRNA and Messenger RNA Detection on Arrays |
| US20110059453A1 (en) * | 2009-08-23 | 2011-03-10 | Affymetrix, Inc. | Poly(A) Tail Length Measurement by PCR |
| WO2013096798A2 (en) | 2011-12-22 | 2013-06-27 | Ibis Biosciences, Inc. | Amplification of a sequence from a ribonucleic acid |
Family Cites Families (4)
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| US4952496A (en) * | 1984-03-30 | 1990-08-28 | Associated Universities, Inc. | Cloning and expression of the gene for bacteriophage T7 RNA polymerase |
| US5194370A (en) * | 1990-05-16 | 1993-03-16 | Life Technologies, Inc. | Promoter ligation activated transcription amplification of nucleic acid sequences |
| US5514545A (en) * | 1992-06-11 | 1996-05-07 | Trustees Of The University Of Pennsylvania | Method for characterizing single cells based on RNA amplification for diagnostics and therapeutics |
| US5723290A (en) * | 1994-11-03 | 1998-03-03 | Trustees Of The University Of Pennsylvania | Methods for profiling mRNA expression in neurites |
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1998
- 1998-03-27 US US09/049,806 patent/US6114152A/en not_active Expired - Fee Related
- 1998-12-14 CA CA002311333A patent/CA2311333A1/en not_active Abandoned
- 1998-12-14 EP EP98964012A patent/EP1038032A1/en not_active Withdrawn
- 1998-12-14 JP JP2000524478A patent/JP2001526053A/en active Pending
- 1998-12-14 WO PCT/US1998/026806 patent/WO1999029907A1/en not_active Application Discontinuation
- 1998-12-14 AU AU19222/99A patent/AU733092B2/en not_active Ceased
Also Published As
| Publication number | Publication date |
|---|---|
| AU733092B2 (en) | 2001-05-03 |
| AU1922299A (en) | 1999-06-28 |
| EP1038032A1 (en) | 2000-09-27 |
| JP2001526053A (en) | 2001-12-18 |
| WO1999029907A1 (en) | 1999-06-17 |
| US6114152A (en) | 2000-09-05 |
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