WO1990001064A1 - Techniques d'amplification selectve de sequences - Google Patents

Techniques d'amplification selectve de sequences Download PDF

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Publication number
WO1990001064A1
WO1990001064A1 PCT/US1989/003099 US8903099W WO9001064A1 WO 1990001064 A1 WO1990001064 A1 WO 1990001064A1 US 8903099 W US8903099 W US 8903099W WO 9001064 A1 WO9001064 A1 WO 9001064A1
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sequence
fragments
primer
fragment
strand
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PCT/US1989/003099
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English (en)
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James Larrick
Kirk Fry
Albert Tam
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Genelabs Incorporated
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Publication of WO1990001064A1 publication Critical patent/WO1990001064A1/fr

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    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C12Q1/683—Hybridisation assays for detection of mutation or polymorphism involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12Q1/6855—Ligating adaptors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12Q1/6858—Allele-specific amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to methods of selectively amplifying sequence specific RNA and single- or double-stranded DNA fragments.
  • RNA or DNA fragments from a fragment mixture. These techniques may be used, for example, to identify the presence of disease-related nucleic acid species in a cellular or tissue sample, or to isolate a specific gene or RNA transcript in mixture of cellular or viral RNA or DNA fragments.
  • One method for selecting a specific sequence from a mixture involves reacting the RNA or DNA species with a complementary-sequence probe which may be bound to a solid support, and/or which carries a label for quantitating probe binding to the nucleic acid fragments.
  • Another method is designed for isolating sequences which are unique to one of two sources of fragment material. In this method, fragments from the two sources are mixed, denatured, reannealed, then separated on the basis of cross-hybridization (common sequences) and self- hybridization (unique sequences). Both of these methods are limited by the amount of fragment material obtainable from the fragment sources, or involve intermediate cloning steps to increase the amount of fragment material.
  • PCR polymerase chain reaction
  • a related object of the invention is to provide such a method which allows sequence specific fragment amplification with a single homopolymeric probe.
  • Still another object of the invention is to provide a method for amplifying the region of a sequence- specific RNA or DNA fragment from a known 3' sequence region to the 5' end of the fragment, such as 5 '-end restriction sites.
  • the method may be used to amplify and identify sequence-specific restriction fragment length polymorphisms.
  • Another object of the invention is to provide such a method which can produce amplified fragments with selected end restriction sites for either directional or random-direction cloning.
  • the invention provides a method of selectively amplifying nucleic acid fragments having a region of known sequence.
  • the method includes mixing sense-strand frag ⁇ ments containing the known sequence with a sequence- specific primer having a 3' sequence which is homologous to the known sequence, and reacting the primed fragments under strand synthesis conditions.
  • the resulting anti- sense strands which have a common primer sequence at their 5' ends, are treated with terminal deoxynucleotide transferase (TdT) and a selected deoxynucleoside triphosphate, to add a homopolymeric sequence to the 3' ends of the anti-sense strand fragments.
  • the fragments a mixed with a homopolymer primer which is homologous to a 3 end homopolymeric sequence in the anti-sense strands, a known- or common-sequence primer which is homologous to t complement of the 5'-end common primer sequence, DNA polym rase, and all four deoxynucleoside triphosphates.
  • T sequence-specific fragments are amplified by repeated cycl of primer annealing, polymerization, and denaturation.
  • the method may be used for amplifying duplex fra ments, such as genomic fragments, having a region of kno sequence.
  • duplex fragments are preferably fir blocked at their 3' ends, prior to initial anti-sense stra formation, to prevent homopolymer tailing of the original D strands.
  • the specific-sequence prim contains a 5' sequence which is complementary to the hom polymeric sequence added at the 3' end of the anti-sen strand, and the homopolymer primer also serves as the commo sequence primer, i.e., only a single primer is required f amplification.
  • the method is also useful for identifying restri tion fragment length polymorphisms in duplex DNA fragment where the fragments of interest have a region with the sa known sequence, and specific restriction endonuclease sit which are spaced at different distances from the kno sequence region. After amplifying the different restricti length fragments, fragment length differences are observed size fractionating the fragments, such as by g electrophoresis.
  • the method may also be used for placing the same different selected restriction endonuclease sites at the en of the amplified fragments, for non-directional or directio fragment cloning.
  • the method is designed for selectively amplifying anti-sense strand fragments derived from single-strand RNA, typically cDNA strands derived from messenger RNA (mRNA) .
  • sequence-specific primer is mixed with the RNA species, to produce an anti-sense strand cDNA which has a common primer sequence at its 5' end.
  • the 5'- end sequence of the specific-sequence primer is com ⁇ plementary to the homopolymeric sequence which is added at the 3' ends of the anti-sense strands, and the homopolymer primer also serves as the defined-sequence primer, i.e., amplification requires only a single primer.
  • the ampli ⁇ fied fragments may be equipped with selected restriction site ends, as above.
  • Figure 1 is a flow diagram of a method of selectively amplifying an RNA species having a region R of known sequence, according to one embodiment of the method of the invention;
  • Figure 2 shows how the amplification method of
  • Figure 1 can be adapted for use in directional cloning of the amplified fragments
  • Figure 3 is a flow diagram of a method of selectively amplifying a duplex DNA fragment having a region R of known sequence, according to another embodiment of the method of the invention.
  • Figure 4 illustrates the use of the fragment amplification method for detecting restriction length ragment polymorphisms.
  • the method of the invention is useful for selectively amplifying DNA fragments which are derived from single-strand RNA species, typically messenger RNA (mRNA) species, having a region of known sequence.
  • RNA species typically messenger RNA (mRNA) species
  • Methods for isolating mRNA species from tissue, cellular or body-fluid samples are well known.
  • One method involves formation of a vanadyl-RNA complex, extraction of protein with chloroform/phenol, and precipitation with cold ethanol.
  • the RNA is extracted from a guanidium isothiocyanate mixture with phenol, followed by a chloroform:isoamyl alcohol extraction, and precipitation of RNA from the aqueous phase with cold ethanol.
  • the reader is referred to Maniati ⁇ , pp. 188-198, and refer ⁇ ences cited therein for details.
  • One preferred RNA isola ⁇ tion method is described in Cathala.
  • RNAs are characterized by a 3' terminal poly A sequence which allows isolation by affinity chromatography, using oligo dT bound to a solid support.
  • total isolated RNA can be further fractionated by density gradient centrifugation, or agarose gel electrophoresis, to obtain a desired size fraction of RNA species.
  • Figure 1 shows a poly A-containing mRNA (poly A RNA) species having a region R of known sequence. This species is typically present as one of a large number of other RNA species with different sequences.
  • the RNA species are mixed with a sequence-specific primer having a region which is homologous to the known sequence in region R.
  • the primer may also include a region which has an unrelated sequence, preferably a homopolymeric sequence which is 5 ' with respect to the sequence-specific primer region, such as a poly dC sequence, as indicated in the figure.
  • the region of the primer which is homologous to the known sequence is indicated by a sawtooth line in the figure, and typically includes at least about 20, and up to several hundred or more specific bases. Where the primer includes an additional homopolymer sequence, such preferably includes at least about 14-20 homopolymer bases.
  • the known-sequence RNA which is primed by the added primer, is copied under standard conditions by reverse transcriptase in the presence of all four deoxynucleoside triphosphates (dNTPs). As seen in Figure 1, this reaction produces an anti-sense cDNA strand which extends from the known-sequence region of the RNA to the 5' RNA end.
  • the RNA/cDNA duplex fragment mixture which also includes single-stranded RNA species, is treated to remove primer and the dNTPs, for example, by one or more phenol or ammonium acetate precipitation steps and gel filtration on Sephacryl S-400 (Pharmacia).
  • the resulting cDNA fragments are reacted with terminal deoxynucleotide transferase (TdT) in the presence of a selected dNTP, to form a homopolymer region at the 3' cDNA ends.
  • TdT terminal deoxynucleotide transferase
  • the homopolymer is poly dG
  • the homopolymer tailing re- action is performed with RNA/cDNA duplex fragments.
  • the efficiency of the reaction can be enhanced by initial digestion with a 5' exonuclease, to expose the 3' fragment ends, or by carrying out the reaction in the presence of cobalt ions (Roychoudhury) .
  • the reaction is carried out under conditions which place at least about 10 and preferably 15 or more bases at the 3' fragments ends.
  • the reaction described in Example 1, which yields poly dG tailing is exemplary. If the tailing reaction is performed with RNA/cDNA fragments, the duplex fragments are now digested with RNase to remove the sense-strand RNA, and the single-strand RNA species in the mixture, yielding single-strand, anti-sense strand cDNAs containing a 5'-end region derived from the specific-sequence primer, the region R of known sequence, and a 3'-end homopolymer tail, e.g., a poly dG tail.
  • the RNase, TdT and selected dNTP are removed, for example, by cDNA fragment precipitation. Since homopolymer tailing by TdT is effective with both single- and double-stranded material, the RNA strands in the RNA/cDNA duplex fragments can be removed with RNase digestion prior to homopolymer tailing.
  • One advantage of homopolymer tailing applied to the mRNA/cDNA fragments is that the tailing method selects for full-copy cDNA fragments. This is because the terminal transferase reaction with duplex fragments requires that the 3'-end of the cDNA be substantially coterminous with or longer than the 5' end of the RNA strand. Thus a partially copied cDNA will underhang the RNA strand and not be tailed ef ⁇ ficiently. As a result, the strand will not be amplified.
  • the specific- sequence anti-sense cDNA strands from above are mixed with a homopolymer primer which is homologous to the 3'-end homopolymer tail of the cDNA strands.
  • a homopolymer primer which is homologous to the 3'-end homopolymer tail of the cDNA strands.
  • the term "homologous” means that the primer is effective to prime by base-pair specific attachment a primer-binding sequence of a .DNA strand, e.g., the 3' homopolymeric region, under the annealing conditions employed.
  • the primer sequence is the exact complement of the primer binding region, although variations in sequence correspondence may be allowed, particularly for longer primer sequences.
  • the primer includes a poly dC region, containing preferably a run of at least about 15 dC bases.
  • the primer may also contain the sequence of a selected restriction endonuclease site, such as an Xhol site which can be used for fragment cloning. It is noted that homopolymer regions generally have more homopolymer subunit ⁇ than actually shown in the figures.
  • initial replication of the anti-sense strands, using the homopolymer primer yields complementary sense strands whose 3' end regions contain a sequence which is complementary to the common sequence provided by the specific-sequence primer.
  • the specific-sequence primer contains a poly dC sequence, as indicated in Figure 1, the sense strand will terminate with a 3'-end poly dG sequence.
  • the amplification mixture further includes a common-sequence primer which is homologous to the complement of the common-sequence at the 5'-end region of the anti-sense strands.
  • the common-sequence primer may be homologous to the known-sequence region of the initial fragment used to form the anti-sense strands, since the sense strands formed from the anti-sense strands will contain this known primer sequence at their 3' ends.
  • the specific-sequence primer includes a 5'-end poly dC sequence which forms the 5' end region of the first-strand (anti-sense) cDNA.
  • the homopolymer which is formed at the 3' end of the anti-sense strand is the complement of the 5'-end sequence, namely poly dG.
  • the complementary homopolymer ends allow the anti-sense strands to be amplified with a single homopolymer primer which is homologous to the 3'-end strand sequence. That is, the homopolymer primer also serves as the common- sequence primer.
  • One preferred homopolymer primer has the following sequence, which includes a 5'-end Xhol site and two protective G bases: d( 5 ' -GGCTCGAGC 20 -3 ' ) Xhol
  • the primer is present in large molar excess with respect to the fragment concentration, and comparable or greater in concentration to the calculated intramolecular strand end concentration, to minimize hybridization between the complementary fragment ends, which can lead to either fragment circles or concatemers.
  • the primer concentration is at least about 10 fold higher than cDNA fragment concentration, and about luM or greater. It may also be necessary to supplement the reaction with added primer during amplification, to replenish primer used up in strand replication.
  • Purified anti-sense cDNA strands from above are amplified by repeated fragment duplication according to the following steps.
  • the fragments are mixed with a large molar excess of the homopolymer primer .described above.
  • the primer sequences and lengths are such as to allow primer attachment to the 3'-end homopolymeric regions of the sense and anti-sense strands, sufficient for DNA polymerase priming.
  • Another requirement is that the primer, when attached to the linker region of the denatured fragment strands, be capable of priming polymerase-catalyzed strand replication; that is, that the internal end of the primer provides a free 3'-OH.
  • the denatured fragments and primer are initially cooled to a temperature, typically between about 37-60°C, "to allow homopolymer primer attachment to the anti-sense cDNA fragments.
  • the four dNTPs and a DNA polymerase capable of catalyzing second-strand primed replication are added, and the reaction mixture is brought to a temperature suitable for enzymatic strand replication.
  • the dNTPs and polymerase were added prior to fragment de- naturation. After heat denaturing, the mixture was an ⁇ nealed at 50°C for two minutes, then brought to 72°C for 5-12 minutes for primed, second-strand replication.
  • the DNA polymerase used was Thermus aquaticus DNA polymerase (Taq DNA polymerase), which is relatively heat-stable at up to 95°C for brief periods.
  • the homopolymer primer hybridizes with the 3 '-end homopolymer regions in each DNA strand, and thus a doubling of fragment number occurs with each replication.
  • the above replication procedure which involves fragment denaturation, annealing to form fragment strand primer complexes, and second strand replication of the complexes in the presence of DNA polymerase, is repeated until a desired concentration of fragments is achieved.
  • the three replication steps are carried out simply by heating the fragment mixture to a denaturing temperature (above the T of the fragments), cooling briefly to allow fragment/primer complex formation, and incubating for a period sufficient for second-strand synthesis.
  • the period of polymerization may be extended for up to about one-half hour for longer fragments, e.g., up to about 8 kilobases in length.
  • a 10 fold amplification can be achieved with about 10 rounds of replication, and a 10 fold amplification with about 20 rounds of replication.
  • initial picogram amounts of sequence-specific RNA species will yield microgram amounts of fragment material after about 20 rounds of replication.
  • Example 1 illustrates the application of the amplification method to a mixture of poly A RNA species isolated from human peripheral T cells.
  • the specific- primer sequence used for initial cDNA synthesis is a human interleukin-2 gene sequence having a 5'-end poly dC sequence.
  • the selectively amplified fragments are separated from the polymerase and polynucleotide components, typically by phenol extraction. If the homopolymer primer used for amplification includes a 5'-end restriction site sequence, the fragments can be cut with the corresponding endonuclease, and cloned into a compatible insertion site in a suitable vector, according to known cloning procedures.
  • Figure 2 illustrates another embodiment of the invention, in which selective amplification results in duplex DNA fragments having different selected restriction sites at opposite fragment ends, to allow directional cloning of the amplified fragments.
  • the method differs from the single-primer method above in that the opposite ends of the anti-sense strands which are amplified have non-complementary sequences.
  • amplification requires two different- sequence primers, i.e., the homopolymer and common- sequence primers have different sequences. These two different primers may be further distinguished by different 5'-end restriction site sequences, to produce different selected restriction sites at the opposite ends of the amplified fragments.
  • the specific-sequence primer used for first-strand cDNA synthesis contains a 5' poly dA sequence which will become the 5'-end common sequence of the anti-sense strand cD As.
  • the fragments are treated with TdT in the presence of a selected deoxynucleoside triphosphate, e.g., dGTP, to form a homopolymer tail, e.g., poly dG, at the first strand cDNA 3' end.
  • a selected deoxynucleoside triphosphate e.g., dGTP
  • a homopolymer tail e.g., poly dG
  • one end of the anti-sense fragment has a poly dA or poly dT sequence, and the opposite end, a poly dC or poly dG sequence.
  • the anti- sense strands are mixed with a homopolymer primer which includes a 5 '-end sequence corresponding to one selected restriction site, and a common-sequence primer which includes a 5'-end sequence corresponding to a second selected restriction site, where the primer restriction sequences preferably correspond to sites not present in the known sequence region of the fragment being amplified.
  • the two primers may further include protective bases at their 5 ' ends-
  • An exemplary homopolymer primer having a 5' Xbal site and a common-sequence primer having a 5' Xhol site are shown at A and B below, respectively:
  • the anti-sense fragments which represent only those original RNA species having the known selected sequence, are amplified as above, with repeated cycles of primer annealing, polymerization, and denaturation. At each cycle, the homopolymer primer is used to prime the anti-sense strand, and the common-sequence primer, the sense strand.
  • the fragments are digested with the two restriction endonucleases formed at the amplified fragment ends, and ligated into a suitable cloning vector which has directional ends which are compatible with the fragment ends.
  • the cloning method described in Example 1 is illustrative.
  • the fragments are formed with rare fragment-end restriction sites Xbal and Xhol, digested with the two restriction enzymes and ligated into a Bluescript- vector having unique Xbal and Xhol sites. Ligation and selection of successful recombinants is conventional.
  • Preferred vectors for directional cloning contain an RNA polymerase promoter site adjacent the insertion site which corresponds to the 5' end of the amplified RNA.
  • this vector is cut at the restriction site at the fragment insert end opposite the promoter, and treated with RNA polymerase in the presence of all four NTPs, the system produces RNA strands which can be used by the host for protein production.
  • the two primer restriction sites must be selected such that insertion into the vector places the 5' end of the anti-sense strand in the duplex cDNA adjacent the polymerase promoter.
  • the cloning vector may be provided with RNA polymerase promoters at both insertion sites, allowing for sense-strand transcription by cutting the recombinant vector at the fragment end which insures sense-strand transcription.
  • the invention is also useful for selective amplification of DNA fragments, typically genomic DNA fragments, containing a region of known sequence.
  • Genomic DNA from a selected cell source can be isolated by standard procedures, which typically include successive phenol and phenol/chloroform extractions with ethanol precipitation.
  • the isolated DNA may be obtained from isolated chromosomes or chromosomal regions of interest.
  • the duplex DNA is fragmented preferably by partial or complete digestion with one or more selected restriction endonucleases, although mechanical shearing may be employed.
  • the fragmented genomic pieces may be size fractionated, or further treated to remove repetitive DNA.
  • Linearized or fragmented plasmid DNA, or fragmented phage DNA is another source of DNA fragments which one might wish to amplify.
  • the vector DNA is obtained from purified plasmid or phage DNA according to conventional techniques, and linearized and/or fragmented by digestion with selected restriction endonuclease(s) .
  • the selective amplification method which is illustrated in Figure 3, generally follows the amplifica ⁇ tion method described above, except that the initial sense-strand template used to form the sequence-specific anti-sense strands is single-stranded DNA rather than RNA.
  • the duplex fragments are first treated to block the 3 '-end OH groups of the duplex fragments. This insures that homopolymer tailing occurs only at the 3'-ends of newly synthesized (with sequence-specific priming) DNA strands. Blocking of the 3' fragment ends may be carried out using T4 DNA polymerase and cordycepin triphosphate (3'-deoxy ATP) using conditions similar to those described in Maniati ⁇ (pp. 118-119) for repair of 3' DNA ends.
  • the initial duplex fragment mixture includes at least a small portion of DNA fragments containing the region R with a known sequence.
  • the duplex fragments are denatured, preferably by heating, and mixed with a sequence-specific primer such as described above contain- ing a 3' region (shown as a sawtooth line in the figure) which is homologous to the known sequence region.
  • the sequence-specific primer also includes a homopolymeric 5' sequence which is homologous to the homopolymer tail to be formed at the 3' ends of the newly made anti-sense strands.
  • the primed fragments are copied with DNA polymerase and all four dNTPs, producing a new DNA strand having the common-specific primer sequence at its 5' end.
  • This new strand is also referred to herein as an anti-sense strand, in analogy to first-strand cDNA synthesis.
  • the fragments are treated with TdT and a selected dNTP, to produce homopolymer tailing at newly synthesized strands in the duplex fragments, as indicated in the figure.
  • the homopolymer tail is preferably complementary to the primer sequence, such as poly dG, allowing amplification with same-sequence primers.
  • Selective amplification of the known-sequence fragments is carried out as above, by repeated cycles of primer annealing, polymerization, and denaturation, until a desired degree of amplification is achieved. The method is illustrated in Example 2.
  • One application of the selective amplification method of the invention is for identifying and/or isolat- ing from a fragment mixture, RNA or DNA species having a known sequence.
  • the method is useful as a diagnostic tool for identifying the presence of an infective or other pathogenic agent in a fragment mixture obtained from a cellular, tissue, or body-fluid source.
  • the nucleic acid fragments are extracted from the source, and those fragments with the selected known sequence are amplified as above.
  • the amplified fragments can be detected by conventional means, such as hybridiza- tion with sequence-specific probes and/or can be isolated and purified, such as by hybridization with sequence- specific biotinylated probes, coupled with binding to an affinity solid support.
  • the method may also be used for cloning specific-sequence RNA or DNA species, particularly where these are present in low amounts and/or represent only a very small portion of the total fragment material.
  • the method exploits the ability to place selected restriction sites at the ends of amplified fragments for directional or non-directional cloning.
  • the method may be used to amplify the coding sequence of a selected mRNA species, from a RNA fragment mixture, and to insert the fragment into a suitable expression vector.
  • FIG. 4 Another application of the invention, for identification and analysis of restriction fragment length polymorphisms (RFLPs), is illustrated in Figure 4.
  • the two DNA fragments at the top in the figure represent genomic DNA fragments which each have a region R of common sequence, but are distinguished by a mutation which has resulted in a new restriction site, indicated by RE(new) , which has the same sequence as existing restriction site RE, but which is significantly closer to the region of known sequence, as indicated.
  • This restriction site polymorphism may, for example, be diagnostic of a given genetic disease, or may be useful as a marker for determining predisposition to a given genetic disease.
  • the fragments are first digested to completion with the restriction endonuclease which cuts at the RE and RE(new) sites, thus producing fragments which terminate at the sites of interest.
  • the fragments are now treated to block 3'-OH groups, as above, denatured and polymerized in the presence of a specific-sequence primer which can.bind to the region R sequence and which contains a common sequence.
  • a specific-sequence primer which can.bind to the region R sequence and which contains a common sequence.
  • the resulting newly synthesized strands extend from a 5'-end primer sequence to the restriction site of interest at the 3' strand ends.
  • Homopolymer tailing now selectively adds a homopolymer sequence to the 3'-ends of the newly synthesized strands.
  • the fragments are amplified, as above, in the presence of same-sequence or different-sequence primers.
  • the strand ends are formed, as above, for same-sequence primer amplification.
  • the sizes of the amplified fragments are now examined, typically by size fractionation on agarose gel and Southern blotting using a radiolabeled probe. The results of an idealized gel pattern are shown at the bot ⁇ tom in the figure. From this gel pattern, it can be determined that a restriction fragment size polymorphism exists, and the sizes of the two fragments can be measured.
  • Terminal deoxynucleotide transferase (calf thymus), alkaline phosphatase (calf intestine), polynucleotide kinase, E. coli DNA polymerase I (Klenow fragment), and SI nuclease are obtained from Boehringer Mannheim Biochemicals (Indianapolis, IN);
  • Xbal, Xhol, T4 DNA ligase and T4 DNA polymerase are obtained from New England Biolabs (Beverly, MA); and streptavidin agarose, from Bethesda Research Labs (Bethesda, MD) .
  • streptavidin agarose from Bethesda Research Labs (Bethesda, MD) .
  • Low-gelling temperature agarose (Sea Plaque) is obtained from FMC (Rockland, ME).
  • Nitro ⁇ cellulose filters are obtained from Schleicher and Schuell (Natick, MA).
  • Synthetic oligonucleotide linkers and primers are prepared using commercially available automated oligonucleotide synthesizers. Alternatively, custom designed synthetic oligonucleotides may be purchased, for example, from Synthetic Genetics (San Diego, CA) . The cDNA synthesis kit and random priming labeling kits were obtained from Boehringer-Mannheim Biochemical (BMB, Indianapolis, IN).
  • Site specific DNA cleavage is performed by treating with the suitable restriction enzyme (or enzymes) under conditions which are generally understood in the art, and the particulars of which are specified by the manufacturer of these, commercially available restriction enzymes. See, e.g., New England Biolabs, Product Catalog.
  • suitable restriction enzyme or enzymes
  • about 1 ug of plasmid or DNA sequence is cleaved by one unit of enzyme in about 20 ul of buffer solution after 1 hr digestion at 37°C; in the examples herein, typically, an excess of restriction enzyme is used to insure complete digestion of the DNA substrate. Incubation times of about one hour to two hours at about 37°C are workable, although variations can be easily tolerated.
  • cleaved fragments After each incubation, protein is removed by extraction with phenol/chloroform, and may be followed by ether extraction, and the nucleic acid re- covered from aqueous fractions by precipitation with ethanol (70%). If desired, size separation of the cleaved fragments may be performed by polyacrylamide gel or agarose gel electrophoresis using standard techniques. A general description of size separations is found in Methods in Enzymoloqy (1980) 65:499-560.
  • Restriction cleaved fragments may be blunt ended by treating with the large fragment of E. coli DNA polymerase I (Klenow reagent) in the presence of the four deoxynucleoside triphosphates (dNTPs) using incubation times of about 15 to 25 min at 20° to 25°C in 50 mM Tris pH 7.6, 50 mM NaCl, 6 mM MgCl 2 , 6 mM DTT and 0.1-1.0 mM dNTPs.
  • the Klenow fragment fills in at 5' single-stranded overhangs in the presence of the four nucleotides.
  • selective repair can be performed by supplying only one of the, or selected, dNTPs within the limitations dictated by the nature of the overhang.
  • the mixture is extracted with phenol/ chloroform and ethanol precipitated.
  • Treatment under ap ⁇ limbate conditions with SI nuclease results in hydrolysis of any single-stranded portions of DNA. In particular, the nicking of 5' hairpins formed on synthesis of cDNA is achieved.
  • Blocking of 3' ends of DNA is carried out using T4 DNA polymerase and cordycepin triphosphate (3'-deoxy ATP) using conditions similar to those described by
  • Ligations are performed in 15-50 ul volumes under the following standard conditions and temperatures: for example, 20 mM Tris-Cl pH 7.5, 10 mM MgCl 2 , 10 mM DTT, 33 mg/ml BSA, 10 mM-50 mM NaCl, and either 40 mM ATP, 0.01-0.02 (Weiss) units T4 DNA ligase at 14°C (for
  • Intermolecular "sticky-end” ligations are usually performed at 33-100 mg/ml total DNA concentrations (5-100 nM total end concentration) .
  • Intermolecular blunt-end ligations are performed at 1 mM total ends concentration.
  • vector constructions employing "vector frag- ments” the vector fragment is commonly treated with bacterial alkaline phosphatase (BAP) or calf intestinal alkaline phosphatase (CIP) in order to remove the 5' phosphate and prevent self-ligation of the vector.
  • BAP bacterial alkaline phosphatase
  • CIP calf intestinal alkaline phosphatase
  • Diges ⁇ tions are conducted at pH 8 in approximately 10 mM Tris- HCl, 1 mM EDTA using about 1 unit per mg of BAP at 60 C for one hour or 1 unit of CIP per mg of vector at 37 C for about one hour-
  • the preparation is extracted with phenol/ chloroform and ethanol precipitated.
  • religation can be prevented in vectors which have been double digested by additional restriction enzyme digestion and separation of the unwanted fragments.
  • RNA preparation 7 isolated from about 10 cells according to standard procedures (Cathula) .
  • the total RNA preparation is fractionated by oligo dT chromatography, also according to known procedures (Maniatis, p. 211), yielding a poly A mRNA preparation.
  • the final preparation is diluted to an RNA concentration of about 1 ug/ml.
  • the mRNA fragments are mixed with about 1 uM final concentration of a primer containing a 5'-region 20- mer sequence of the human interleukin-2 gene corresponding to the coding region bases 381-400 of exon 1 (Holbrook) , and an 18-mer 3'-region poly dC sequence.
  • the primer is prepared by standard oligonucleotide synthesis and has the sequence:
  • the fragment/primer mixture is converted to first-strand cDNA, using conditions described in the cDNA kit supplied by Boehringer-Mannheim.
  • RNA/cDNA complex is purified by sequential extraction with one volume of phenol/chloroform followed by 2 ethanol (5 ug of tRNA is added as carrier) precipita ⁇ tions using 0-5 volume of 7.5 M NH.OAc pH 7.0 and 2 volumes of ethanol. After centrifugation (10 min in microfuge at room temperature; about 14K rpm) the pellet is completely desiccated in a speed vac to assure complete removal of the NH.OAc which is known to inhibit TdT.
  • the fragment mixture is resuspended in 0.050 ml of 0.2 M potassium cacodylate (pH 7.2), 4 mM MgCl-, 1 mM 2-mercaptoethanol, as provided by BRL, which contains a final concentration of 25 uM of dGTP.
  • BRL potassium cacodylate
  • 15 units of TdT supplied by BRL
  • the reaction is incubated for 30 min at 37°C.
  • ug of RNase A supplied by Boehringer-Mannheim is added to the reaction and incubated at 37 C for an additional 30 min.
  • the reaction mix is extracted with phenol/chloroform as above and 5-10 ug of tRNA is added as carrier before ethanol precipitation, as described above was carried out.
  • the sample may be precipitated without carrier and treated with RNase A after resuspending the samples.
  • Tris-Cl buffer pH 8.3, containing 1.5 mM MgCl2 (Buffer A) is added 100 ul of the above cDNA fragment mixture 2 uM of a primer having the sequence d(5'-GGCTCGAGC 2() -3' ) , 200 uM (final concentra ⁇ tion) each of dATP, dCTP, dGTP, and dTTP, and 5 units of Thermus aquaticus DNA polymerase (Taq polymerase).
  • Buffer A 100 ul of the above cDNA fragment mixture 2 uM of a primer having the sequence d(5'-GGCTCGAGC 2() -3' ) , 200 uM (final concentra ⁇ tion) each of dATP, dCTP, dGTP, and dTTP, and 5 units of Thermus aquaticus DNA polymerase (Taq polymerase).
  • the reaction mixture is heated to 94°C for 1 minute for de- naturation, allowed to cool to 50°C for 2 min for primer annealing, and then heated to 72°C for 5-12 min to allow for primer extension by Taq polymerase.
  • the replication reaction involving successive heating, cooling, and polymerase reaction, was repeated an additional 25 times with the aid of a Perkin Elmer Cetus DNA thermal cycler.
  • the amplified fragments from the sample mixture corresponding to 10 cells are digested with Xhol.
  • Bluescript- M13 plasmid is treated with the same enzyme, and treated with alkaline phosphatase, prior to mixing with the above amplified fragments. Ligation is performed under conditions which promote circularization of the single plasmid fragments. The circularized plasmid is selected on E. coli strain DH5, and successful re- combinant ⁇ are selected for ampicillin resistance.

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Abstract

Un procédé d'amplification sélective de fragments d'acides nucléiques ayant une séquence connue. Des fragments du brin codeur qui contiennent la séquence connue sont amorcés avec une amorce spécifique à la séquence et ayant une séquence homologue de la séquence connue, puis reproduits en présence de polymérase d'ADN afin de former des brins non codeurs ayant la séquence initiatrice à leurs extrémités 5'. On traite les brins non codeurs avec de la transférase de déoxynucléotide terminal et un triphosphate de déoxynucléoside sélectionné afin d'ajouter une séquence homopolymère aux extrémités 3' des brins. On mélange les fragments du brin non codeur ainsi obtenus avec une amorce homopolymère homologue de ladite séquence de fragment homopolymère, avec un initiateur commun à diverses séquences qui est homologue d'une région de l'amorce de séquence spécifique, avec de la polymérase d'ADN et avec les quatre triphosphates de déoxynucléoside. Les fragments ainsi synthétisés sont amplifiés pendant des cycles répétés de recuit dénaturant et de polymérisation amorcée. L'invention porte également sur un procédé qui permet d'identifier et d'analyser des polymorphismes de longueur des fragments de restriction, basé sur ledit procédé d'amplification de fragments.
PCT/US1989/003099 1988-07-26 1989-07-18 Techniques d'amplification selectve de sequences WO1990001064A1 (fr)

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990009457A2 (fr) * 1989-02-14 1990-08-23 Biogen, Inc. Paires d'amorce d'oligonucleotides pour l'amplification genetique independante de la sequence et leurs procedes d'utilisation
EP0567587A1 (fr) * 1991-01-18 1993-11-03 Beth Israel Hospital Association Synthese d'adn entier double brin a partir d'une matrice d'adn lineraire simple brin
WO1994029443A1 (fr) * 1993-06-11 1994-12-22 Mueller Manfred W Analyse de sequences d'arn par pcr
US5422252A (en) * 1993-06-04 1995-06-06 Becton, Dickinson And Company Simultaneous amplification of multiple targets
EP0718409A2 (fr) * 1994-12-22 1996-06-26 Hitachi, Ltd. Méthode de préparation d'ADN
EP0767240A2 (fr) * 1995-09-18 1997-04-09 Hitachi, Ltd. Procédé de séquencage et de préparation d'ADN
EP0778351A3 (fr) * 1995-11-30 1999-01-20 Hitachi, Ltd. Méthode d'analyse ou essai de polynucléotides et d'analyseur ou instrument de polynucléotides
US5990300A (en) * 1994-09-02 1999-11-23 Andrew C. Hiatt Enzyme catalyzed template-independent creation of phosphodiester bonds using protected nucleotides
US6214987B1 (en) 1994-09-02 2001-04-10 Andrew C. Hiatt Compositions for enzyme catalyzed template-independent formation of phosphodiester bonds using protected nucleotides
US6232465B1 (en) 1994-09-02 2001-05-15 Andrew C. Hiatt Compositions for enzyme catalyzed template-independent creation of phosphodiester bonds using protected nucleotides

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4683194A (en) * 1984-05-29 1987-07-28 Cetus Corporation Method for detection of polymorphic restriction sites and nucleic acid sequences
US4683202A (en) * 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US4683195A (en) * 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
US4800159A (en) * 1986-02-07 1989-01-24 Cetus Corporation Process for amplifying, detecting, and/or cloning nucleic acid sequences

Patent Citations (6)

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Publication number Priority date Publication date Assignee Title
US4683194A (en) * 1984-05-29 1987-07-28 Cetus Corporation Method for detection of polymorphic restriction sites and nucleic acid sequences
US4683202A (en) * 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US4683202B1 (fr) * 1985-03-28 1990-11-27 Cetus Corp
US4683195A (en) * 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
US4683195B1 (fr) * 1986-01-30 1990-11-27 Cetus Corp
US4800159A (en) * 1986-02-07 1989-01-24 Cetus Corporation Process for amplifying, detecting, and/or cloning nucleic acid sequences

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* Cited by examiner, † Cited by third party
Title
MOLECULAR CLONING: A Laboratory Manual, MANIATIS, Cold Spring Harbor Lab., 1982, pages 213, 217, 222 and 374-375. *
SCIENCE, Vol. 243, 1989, LOH, "Polymerase chain reaction with single-sided specificity: analysis of T cell receptor sigma chain", pages 217-218. *

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990009457A2 (fr) * 1989-02-14 1990-08-23 Biogen, Inc. Paires d'amorce d'oligonucleotides pour l'amplification genetique independante de la sequence et leurs procedes d'utilisation
WO1990009457A3 (fr) * 1989-02-14 1990-10-04 Biogen Inc Paires d'amorce d'oligonucleotides pour l'amplification genetique independante de la sequence et leurs procedes d'utilisation
EP0567587A1 (fr) * 1991-01-18 1993-11-03 Beth Israel Hospital Association Synthese d'adn entier double brin a partir d'une matrice d'adn lineraire simple brin
EP0567587A4 (en) * 1991-01-18 1997-05-21 Beth Israel Hospital Synthesis of full-length, double-stranded dna from a single-stranded linear dna template
US5422252A (en) * 1993-06-04 1995-06-06 Becton, Dickinson And Company Simultaneous amplification of multiple targets
WO1994029443A1 (fr) * 1993-06-11 1994-12-22 Mueller Manfred W Analyse de sequences d'arn par pcr
US6232465B1 (en) 1994-09-02 2001-05-15 Andrew C. Hiatt Compositions for enzyme catalyzed template-independent creation of phosphodiester bonds using protected nucleotides
US6214987B1 (en) 1994-09-02 2001-04-10 Andrew C. Hiatt Compositions for enzyme catalyzed template-independent formation of phosphodiester bonds using protected nucleotides
US5990300A (en) * 1994-09-02 1999-11-23 Andrew C. Hiatt Enzyme catalyzed template-independent creation of phosphodiester bonds using protected nucleotides
EP0718409A3 (fr) * 1994-12-22 1999-02-03 Hitachi, Ltd. Méthode de préparation d'ADN
EP0718409A2 (fr) * 1994-12-22 1996-06-26 Hitachi, Ltd. Méthode de préparation d'ADN
EP0767240A3 (fr) * 1995-09-18 1999-12-22 Hitachi, Ltd. Procédé de séquencage et de préparation d'ADN
EP0767240A2 (fr) * 1995-09-18 1997-04-09 Hitachi, Ltd. Procédé de séquencage et de préparation d'ADN
EP0778351A3 (fr) * 1995-11-30 1999-01-20 Hitachi, Ltd. Méthode d'analyse ou essai de polynucléotides et d'analyseur ou instrument de polynucléotides

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