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WO2016013620A1 - Procédé d'amplification d'adn à l'aide d'arn hélicase à boîte dead dérivée d'archée thermophile ou d'un variant de celle-ci, et arn hélicase à boîte dead dérivée d'archée thermophile et variant de celle-ci à utiliser pour un procédé d'amplification d'adn - Google Patents

Procédé d'amplification d'adn à l'aide d'arn hélicase à boîte dead dérivée d'archée thermophile ou d'un variant de celle-ci, et arn hélicase à boîte dead dérivée d'archée thermophile et variant de celle-ci à utiliser pour un procédé d'amplification d'adn Download PDF

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WO2016013620A1
WO2016013620A1 PCT/JP2015/070980 JP2015070980W WO2016013620A1 WO 2016013620 A1 WO2016013620 A1 WO 2016013620A1 JP 2015070980 W JP2015070980 W JP 2015070980W WO 2016013620 A1 WO2016013620 A1 WO 2016013620A1
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dead
variant
rna helicase
derived
pcr
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藤原　伸介
綾子 藤原
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Kwansei Gakuin Educational Foundation
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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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • 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
    • 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

Definitions

  • the present invention relates to a DNA amplification method using a thermophilic archaea-derived DEAD-box type RNA helicase and a variant thereof, and a thermophilic archaea-derived DEAD-box type RNA helicase and a variant thereof used for the method.
  • a DNA amplification method by polymerase chain reaction (PCR) is widely used.
  • the PCR method has a problem that a sequence different from a desired DNA sequence is erroneously amplified.
  • PCR is carried out using two pairs of primer sets that target both ends of the base sequence to be amplified and have complementary sequences thereto.
  • Non-specific amplification occurs when the primer misanneals with a base sequence other than the target.
  • misannealing is likely to occur when the target base sequence is a palindromic sequence or has a high GC content.
  • the primer dimer is introduced between the preparation of the PCR reaction solution containing the DNA polymerase and the primer and the start of PCR.
  • side reactions such as degradation of the primer proceed. This is known to cause nonspecific amplification.
  • Non-Patent Documents 1 to 3 Conventionally, as a means for suppressing such misamplification, a primer design has been devised or an annealing temperature has been improved. In addition, when using a DNA polymerase having proofreading activity, these activities are suppressed at room temperature by binding two kinds of neutralizing antibodies against the polymerase activity and the proofreading activity to the DNA polymerase. It has also been performed to suppress side reactions that can cause amplification (Non-Patent Documents 1 to 3).
  • An object of the present invention is to provide means for suppressing nonspecific amplification in PCR.
  • thermophilic archaia-derived DEAD-box RNA helicase The inventors of the present invention have intensively studied to solve the above problems and completed the present invention. Specifically, the present inventors have found that nonspecific amplification can be suppressed by performing PCR in the presence of a thermophilic archaia-derived DEAD-box RNA helicase. The present inventors have conducted further studies and have found that such effects are derived from heat resistance and helicase activity among the functions and properties of thermophilic archaia-derived DEAD-box RNA helicase.
  • Item 1 A DNA amplification method by polymerase chain reaction, A method comprising a step of performing a polymerase chain reaction in the presence of a thermophilic archaea-derived DEAD-box RNA helicase or a variant thereof having heat resistance and helicase activity.
  • Item 2. Item 8. The method according to Item 1, wherein the thermophilic archaea-derived DEAD-box RNA helicase comprises the amino acid sequence represented by any one of SEQ ID NOs: 1 to 7.
  • Item 3. Item 3.
  • Item 2 wherein the variant comprises an amino acid sequence having 80% or more homology with the amino acid sequence represented by any of SEQ ID NOs: 1 to 8.
  • Item 4. Item 4. The method according to any one of Items 1 to 3, wherein the variant exhibits helicase activity at 90 ° C.
  • Item 5. The method according to any one of Items 1 to 4, wherein the variant exhibits unwinding activity of a double-stranded nucleic acid at 50 ° C and ATPase activity in the presence of the double-stranded nucleic acid at 90 ° C.
  • Item 6. Item 6.
  • thermophilic archaea-derived DEAD-box type RNA helicase The method according to any one of Items 1 to 5, wherein the step is a polymerase chain reaction in the presence of 20 to 150 nM of the thermophilic archaea-derived DEAD-box type RNA helicase.
  • Item 7. The method according to any one of Items 1 to 6, wherein the step is a step of performing a polymerase chain reaction in the presence of magnesium ions.
  • FIG. 1 is a drawing schematically showing an action mechanism in which non-specific proliferation in PCR is suppressed by the action of a thermophilic archaea-derived DEAD-box type RNA helicase or a variant thereof used in the present invention. It is drawing which shows the measurement result of the ATPase activity of an Example. It is drawing which shows the evaluation result of the influence on PCR amplification by SF2 helicase addition of an Example. Is a view showing verification results of PCR efficiency due to the high concentration MgSO 4 addition Example. It is drawing which shows the result of the nonspecific band disappearance test by TK0566 addition of an Example.
  • Thermophilic archaea-derived DEAD-box RNA helicase has a characteristic motif of Asp-Glu-Ala-Asp and exhibits RNA helicase activity.
  • thermophilic archaia-derived DEAD-box RNA helicase used in the present invention is derived from a thermophilic archaea, and thus exhibits helicase activity even in a high-temperature environment. Although it does not specifically limit, Preferably helicase activity is shown in 90 degreeC.
  • the thermophilic archaea-derived DEAD-box RNA helicase used in the present invention is preferably derived from a hyperthermophilic archaea.
  • thermophilic archaic DEAD-box RNA helicase used in the present invention are not particularly limited.
  • it is derived from Thermococcus kodakarensis consisting of the amino acid sequence represented by SEQ ID NO: 1. TK0566.
  • TK0566 ortholog can be used instead of TK0566.
  • the TK0566 ortholog is a DEAD-box type RNA helicase and has the same function as TK0566.
  • TK0566 ortholog is considered to be an archaea-specific functional molecule from the results of Bootstrap analysis of the phylogenetic tree.
  • it is important that the RNA helicase used is not inactivated by PCR. Therefore, in the present invention, a DEAD-box RNA helicase derived from a thermophilic archaea can be used.
  • TK0566 orthologs have been found to be present in particular at the gates of Euryarchaota and Clenarchaeota. It is known that the TK0566 ortholog belonging to the Yuriakiota gate shows 77% coverage and 43% minimum (identity) by BLAST (registered trademark) analysis as compared with TK0566. Further, it has been found that the TK0566 ortholog belonging to the Clenarchiota Gate shows a cover rate of 40% and a minimum value (identity) of 27% by BLAST (registered trademark) analysis as compared with TK0566. Therefore, in the present invention, a DEAD-box type RNA helicase derived from a thermophilic bacterium belonging to the urea chiota gate and the clenarchiota gate can be used.
  • thermophile belonging to the Yuriakiota gate is not particularly limited. Examples include those belonging to the genus Methanococcus, the genus Methanococcus, and the like.
  • DEAD-box RNA helicase derived from thermophilic bacteria belonging to the genus Thermococcus include, but are not limited to, those derived from Thermococcus kodakarensis (SEQ ID NO: 1) and the like.
  • thermophilic DEAD-box RNA helicase belonging to the genus Pyrococcus include, but are not limited to, those derived from Pyrococcus furiosus (SEQ ID NO: 2) and the like.
  • thermophilic bacterium-derived DEAD-box RNA helicase belonging to the genus Methanothermus include, but are not limited to, those derived from Methanothermus fervidus (SEQ ID NO: 3) and the like.
  • thermophilic bacterium-derived DEAD-box RNA helicase belonging to the genus Methanothermobacter include, but are not particularly limited to, those derived from Methanothermobacter marburgens (SEQ ID NO: 4).
  • thermophilic bacterium-derived DEAD-box RNA helicase belonging to the genus Methanococcus include, but are not particularly limited to, those derived from Methanococcus voltae (SEQ ID NO: 5).
  • thermophilic bacterium-derived DEAD-box RNA helicase belonging to the genus Methanocadococcus include, but are not particularly limited to, those derived from Methanocardococcus jannaschii (SEQ ID NO: 6).
  • thermophilic bacterium belonging to the Clenarchiota is not particularly limited, and examples thereof include those belonging to the genus Pyrobaculum and the genus Sulfolobus.
  • thermophilic bacterium-derived DEAD-box RNA helicase belonging to the genus Pyrobaculum include, but are not limited to, those derived from Pyrobaculum califontis (SEQ ID NO: 7) and the like. .
  • DEAD-box RNA helicases derived from thermophiles belonging to the genus Sulfolobus include, but are not limited to, those derived from Sulfolobus tokodaii (SEQ ID NO: 8) and the like.
  • thermophilic archaic-derived DEAD-box RNA helicase variant which has heat resistance and helicase activity, is also used. it can.
  • the above-mentioned variant is not particularly limited, but preferably exhibits helicase activity at 90 ° C.
  • the above-mentioned variant is not particularly limited, but preferably exhibits the unwinding activity of the double-stranded nucleic acid at 50 ° C. and the ATPase activity in the presence of the double-stranded nucleic acid at 90 ° C.
  • the unwinding activity of the double-stranded nucleic acid is measured as follows.
  • a radiolabeled substrate is used for measuring the unwinding activity (Table 1).
  • 50 pmol of 63merRNA (ssRNA63) was reacted with [ ⁇ - 32 P] ATP (6000 Ci / mmol) (ParkinElmer, Boston, USA) and T4 polynucleotide kinase (Takara bio) at 37 ° C. for 30 minutes and labeled for 32 minutes. To do. Unreacted ⁇ -ATP is removed using Microspin G-25 (GE Healthcare). 20 pmol of ssRNA33, ssRNA48-5 ′, and ssRNA48-3 ′ were added to 10 pmol of labeled ssRNA63, respectively, and treated at 95 ° C.
  • both-end protruding dsRNA, 5 ′ protruding dsRNA, and 3 ′ protruding dsRNA are prepared. This is subjected to Microspin S-400HR to remove RNA that has not been annealed. The obtained dsRNA and unannealed ssRNA are confirmed using a 7% non-denaturing acrylamide gel. The unwinding activity is measured using this ssRNA and the prepared dsRNA as a substrate.
  • the rewinding activity is measured as follows. 10 ⁇ L of a reaction solution containing 32 P-labeled nucleic acid substrate, 0.2 ⁇ M purified protein, 1 mM ATP, 2 mM MgCl 2 , 2 mM DTT, 4 units of RNase inhibitor, 50 mM HEPES (pH 7.6) is reacted at 50 ° C. for 30 minutes. Immediately afterwards, stop the reaction by transferring to ice. The reaction product is applied to a 7% acrylamide gel (NATIVE-PAGE) (Table 2). After drying the gel, the radioactivity of the reaction product is detected by BAS-2500 (FUJIFILM) and exposed to X-ray film.
  • ATPase activity in the presence of double-stranded nucleic acid is measured as follows.
  • ATPase activity is measured by measuring the amount of free phosphate released when ATP is converted to ADP.
  • ssRNA63 SEQ ID NO: 9
  • the total amount of the reaction solution is 10 ⁇ L, and contains 5 nM nucleic acid substrate, 0.2 ⁇ M purified protein, 1 mM ATP, 2 mM MgCl 2 , 2 mM DTT, 4 units of Ribonuclease inhibitor (Humanplacenta) (Takarabio), 50 mM HEPES (pH 7.6). This is reacted at 90 ° C. for 30 minutes and immediately transferred to ice to stop the reaction.
  • the amount of free phosphoric acid in the reaction solution is measured using BIOMOLGREEN TM (BIOMOL), and Ab 530 is measured using Multiskan Spectrum (ThermoLabsystems).
  • the variant is not particularly limited, and consists of an amino acid sequence in which 1 to 100 amino acids are deleted, substituted, or added in TK0566 or TK0566 ortholog consisting of the amino acid sequence represented by any of SEQ ID NOs: 1 to 8. It may be a thing.
  • the variant is preferably 1 to 50, more preferably 1 to 20, more preferably 1 to 10, in TK0566 or TK0566 ortholog consisting of the amino acid sequence represented by any of SEQ ID NOs: 1 to 8, Most preferably, it consists of an amino acid sequence in which one to several amino acids are deleted, substituted or added.
  • the modified body may alternatively be composed of an amino acid sequence having a homology of 80% or more with TK0566 or TK0566 ortholog comprising the amino acid sequence represented by any of SEQ ID NOs: 1 to 8.
  • Said variant is a TK0566 or TK0566 ortholog consisting of the amino acid sequence represented by any one of SEQ ID NOs: 1 to 8, more preferably an amino acid sequence having a homology of 90% or more, more preferably a homology Consists of 95% or more amino acid sequence.
  • TK0566 ortholog is SF2 helicase, and its structural analysis has already been done. It can be roughly divided into an N-terminal region, a helicase core and a C-terminal region from the N-terminal side, and the helicase core has a structure in which domain 1 and domain 2 are connected in this order from the N-terminal side. The DEAD motif is present in domain 1.
  • higher-order structural analysis has also been performed.
  • the modified body can be prepared. Specifically, the above-mentioned variant is introduced by introducing amino acid alterations in other regions as necessary while preserving the regions important for maintaining heat resistance and helicase activity or minimizing the degree of modification. Should be created.
  • homology is determined by comparing two optimally aligned sequences.
  • a method for aligning and comparing the sequences for example, an algorithm of Basic Local Alignment Search Tool (BLAST) can be used.
  • BLAST Basic Local Alignment Search Tool
  • homology in the present invention refers to “identity” in BLAST.
  • the homology may be calculated using another algorithm.
  • DNA amplification method by PCR comprises: It is a method characterized in that PCR is carried out in the presence of a thermophilic archaic-derived DEAD-box RNA helicase or a variant thereof having heat resistance and helicase activity.
  • Nonspecific amplification can be suppressed by the action of a thermophilic archaea-derived DEAD-box type RNA helicase or a modified form thereof.
  • Such effects are derived from heat resistance and helicase activity among the functions and properties of thermophilic archaea-derived DEAD-box RNA helicase.
  • thermophilic archaia-derived DEAD-box RNA helicase or a variant thereof does not lose its helicase activity in PCR due to its heat resistance.
  • thermophilic archaia-derived DEAD-box RNA helicase or a modified product thereof was obtained by non-specifically annealing the primer to the nucleic acid due to its helicase activity, as schematically shown in FIG. Unwind the nucleic acid portion of the strand. This function suppresses nonspecific DNA growth.
  • PCR is not particularly limited and can be performed under a wide range of conditions.
  • thermophilic archaea-derived DEAD-box type RNA helicase or a modified form thereof it is preferable to perform PCR in the presence of 20 to 150 nM thermophilic archaea-derived DEAD-box type RNA helicase or a modified form thereof.
  • PCR is performed in the presence of 20 nM or more thermophilic archaic DEAD-box type RNA helicase or a variant thereof, non-specific amplification can be effectively suppressed.
  • thermophilic archaic-derived DEAD-box type RNA helicase of 150 nM or less or a modified form thereof the amount of specifically amplified DNA is improved.
  • the polymerase chain reaction is preferable to perform the polymerase chain reaction in the presence of magnesium ions in terms of the effects of the present invention.
  • the polymerase chain reaction is more preferably carried out in the presence of 0.5 mM to 3 mM magnesium ions, although not particularly limited.
  • magnesium ions are not particularly limited to the reaction system, but for example, magnesium ions may be added as magnesium sulfate.
  • composition used for DNA amplification method by PCR contains a thermophilic archaea-derived DEAD-box type RNA helicase, or a variant thereof having heat resistance and helicase activity.
  • composition of the present invention further includes, for example, a buffer such as Tris-HCl, a salt such as KCl, a redox agent such as dithiothreitol, a chelating agent such as ethylenediaminetetraacetic acid (EDTA), glycerol, It may contain at least one selected from the group consisting of surfactants such as Tween-20 and NP-40.
  • a buffer such as Tris-HCl
  • a salt such as KCl
  • a redox agent such as dithiothreitol
  • a chelating agent such as ethylenediaminetetraacetic acid (EDTA), glycerol
  • TK0566 Using the total DNA of T. kodakarensis as a template, the TK0566 gene was amplified using primers (TK0566EX-F (SEQ ID NO: 10), TK0566EX-R (SEQ ID NO: 11)). The obtained DNA fragment containing the TK0566 gene was cleaved with NdeI and EcoRI and inserted into pET28a. The constructed pET-TK0566 plasmid was used to transform E. coli BL21-Codon-Plus (DE3) -RIL.
  • IPTG ⁇ -D-1-thiogalactopyranoside
  • the LB medium contains 20 ⁇ g / mL kanamycin and 30 ⁇ g / mL chloramphenicol.
  • the cultured cells were collected, suspended in 10 mL of buffer D: 20 mM Tris-HCl, 500 mM NaCl, 0.1% Triton X-100, pH 7.9, and sonicated.
  • the disrupted solution was centrifuged at 8000 g for 10 minutes, and the supernatant was heat-treated at 80 ° C. for 15 minutes. Then, the supernatant was collected again by centrifugation at 8000 g for 10 minutes. Since His6 tag was added to the N-terminal side of expressed TK0566, it was purified using a Ni column.
  • the column was equilibrated with buffer D, and the supernatant was applied thereto. Then, after washing with buffer E: 20 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, 0.1% Triton X-100, pH 7.9, buffer F: 20 mM Tris-HCl, 500 mM NaCl, 250 mM imidazole, 0.1 Elute with% Triton X-100, pH 7.9. The eluted fraction was dialyzed against buffer D.
  • ATPase activity was measured by measuring the amount of free phosphate released when ATP was converted to ADP.
  • ssRNA63 63mer RNA
  • the total volume of the reaction solution is 10 ⁇ L, and 5 nM nucleic acid substrate, 0.2 ⁇ M purified protein, 1 mM ATP, 2 mM MgCl 2 , 2 mM DTT, 4 units of Ribonuclease inhibitor (Human placenta) (Takara bio), 50 mM HEPES (pH 7.6) Including. This was reacted at 50 ° C. to 110 ° C.
  • FIG. 2 shows the measurement results.
  • 16S rDNA was amplified by PCR using primers (TKr05-Fw (SEQ ID NO: 12) and TKr05-Rv (SEQ ID NO: 13)). Further, the toxA gene was amplified using the total DNA genome of P. aeruginosa as a template and using primers (PA1148-Fw (SEQ ID NO: 14), PA1148-Rv (SEQ ID NO: 15)). The obtained amplified fragment was gel-extracted and purified using Nucleo Spin Gel and PCR Clean-up (Takara). The distributor of PAO1 strain genomic DNA is NBRC, NITE, Kisarazu, Japan (NBRC Number: 106052G).
  • FIG. 3 shows the results of PCR under the following PCR reaction composition (Table 3) and PCR conditions (Table 4) in order to verify whether the purified SF2 helicase affects PCR amplification. As shown in FIG. 3, when TK0566 was added, the effect of reducing PCR misamplification was observed.
  • TK0566 can suppress primer misannealing.
  • TK0566 may have increased the specificity of PCR amplification by specifically interacting with KOD polymerase.
  • KOD polymerase thermophilus
  • ALDH2 is an enzyme that oxidizes aldehydes produced by the metabolism of ethanol, and the lack of activity is due to the point mutation E487K of the ALDH2 gene.
  • genes from combinations of alleles normal homozygotes (NN type), heterozygotes (NM type), and mutant homozygotes (MM type). Sensitivity to alcohol can be determined by examining which of these genotypes are present.
  • the PCR reaction composition and conditions are shown below, and the results are shown in FIG.

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Abstract

La présente invention vise à procurer un moyen d'inhiber l'amplification non spécifique dans une réaction en chaîne par polymérase (PCR). L'invention concerne un procédé d'amplification d'ADN par le biais d'une réaction en chaîne par polymérase, caractérisé en ce que la réaction en chaîne par polymérase est effectuée en présence d'ARN hélicase à boîte DEAD dérivée d'une archée thermophile ou d'un variant de celle-ci qui possède des propriétés thermophiles et une activité hélicase.
PCT/JP2015/070980 2014-07-24 2015-07-23 Procédé d'amplification d'adn à l'aide d'arn hélicase à boîte dead dérivée d'archée thermophile ou d'un variant de celle-ci, et arn hélicase à boîte dead dérivée d'archée thermophile et variant de celle-ci à utiliser pour un procédé d'amplification d'adn Ceased WO2016013620A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2018014924A (ja) * 2016-07-27 2018-02-01 国立大学法人京都大学 ヘリカーゼを用いたｐｃｒ
CN108624640A (zh) * 2017-03-15 2018-10-09 中国科学院微生物研究所 一种利用嗜热引发酶扩增dna的方法

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JP2003506048A (ja) * 1999-07-30 2003-02-18 ストラタジーン 古細菌複製補助因子及び使用方法
JP2006500028A (ja) * 2002-09-20 2006-01-05 ニュー・イングランド・バイオラブズ・インコーポレイティッド 核酸のヘリカーゼ依存性増幅
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JP2003506048A (ja) * 1999-07-30 2003-02-18 ストラタジーン 古細菌複製補助因子及び使用方法
JP2006500028A (ja) * 2002-09-20 2006-01-05 ニュー・イングランド・バイオラブズ・インコーポレイティッド 核酸のヘリカーゼ依存性増幅
JP2012516155A (ja) * 2009-01-27 2012-07-19 キアゲン ゲーザーズバーグ エンドポイント均一蛍光検出を用いた好熱性ヘリカーゼ依存性増幅技術

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AYAKO FUJIWARA ET AL.: "Cho Konetsusei Archaea Yurai Helicase o Mochiita PCR Hanno Tokuisei no Zokyo", NIPPON ARCHAEA KENKYUKAI DAI 27 KAI KOENKAI YOSHISHU, 25 July 2014 (2014-07-25), Retrieved from the Internet <URL:http://archaea.kenkyuukai.jp/images/sys%5Cinformation%5C20150416202844-41B7A09859C5FA49985168C6BA63F22A50C8F0787F1A08E330AC601DA16ABB58.pdf> [retrieved on 20150901] *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2018014924A (ja) * 2016-07-27 2018-02-01 国立大学法人京都大学 ヘリカーゼを用いたｐｃｒ
CN108624640A (zh) * 2017-03-15 2018-10-09 中国科学院微生物研究所 一种利用嗜热引发酶扩增dna的方法
CN108624640B (zh) * 2017-03-15 2021-09-21 中国科学院微生物研究所 一种利用嗜热引发酶扩增dna的方法

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