WO2006098428A1 - Methode pour diminuer l’amplification non-specifique d’une amorce de dimere - Google Patents

Methode pour diminuer l’amplification non-specifique d’une amorce de dimere Download PDF

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Publication number
WO2006098428A1
WO2006098428A1 PCT/JP2006/305372 JP2006305372W WO2006098428A1 WO 2006098428 A1 WO2006098428 A1 WO 2006098428A1 JP 2006305372 W JP2006305372 W JP 2006305372W WO 2006098428 A1 WO2006098428 A1 WO 2006098428A1
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WIPO (PCT)
Prior art keywords
primer
substance
nucleic acid
sequence
binds
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PCT/JP2006/305372
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English (en)
Japanese (ja)
Inventor
Yoshihide Hayashizaki
Yasumasa Mitani
Chiaki Kato
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Kabushiki Kaisha Dnaform
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Publication of WO2006098428A1 publication Critical patent/WO2006098428A1/fr

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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
    • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction

Definitions

  • the present invention relates to a method for improving a nucleic acid amplification method, and more particularly to a method for reducing non-specific amplification of a primer dimarker formed during nucleic acid amplification.
  • Nucleic acid amplification techniques such as PCR have various known powers.
  • Primers are commonly used to form primer dimers in which all primers are paired.
  • primer dimer acts as a cage DNA and a non-specific amplification product is produced. This is because primers are consumed in non-specific amplification reaction, which is not only the problem that other DNA fragments are mixed into the original DNA product, and the efficiency of the target DNA amplification is reduced.
  • Primer design needs to take into account various factors such as oligonucleotide length, Tm value, physical sequence characteristics, and possible primer interactions. This alone completely solves the problem of primer dimers. I can't.
  • the non-specific amplification product and the target DNA product are separated by electrophoresis, and the target amplification product band is cut out or amplified.
  • detection methods capable of distinguishing between non-specific amplification products and target products associated with primer dimer formation have also been developed.
  • a detection method that can distinguish between fluorescence derived from the target product and fluorescence caused by non-specific binding such as primer dimer by continuous monitoring.
  • the dye QSY7 or QSY9 is bound to the 5 'end of the primer, and the fluores such as SYBR Green bound to the primer dimer. Primers designed to quench fluorescence near the mouth fore have also been developed.
  • the bound nucleic acid dye molecule such as SYBR Green fluoresces away from the quencher, but it is short like a primer dimer, and the strand is fluorescent by the quencher. Therefore, a signal of only the target amplification product can be obtained.
  • a primer-dimer is more likely to form non-specific nucleic acid amplification because a DNA-dimer is more likely to be formed at low temperatures, and DNA polymerases usually have some activity even at low temperatures.
  • a hot start method can be mentioned. This is a method in which the reaction of DNA polymerase occurs only after raising the temperature of the reaction solution. Specific examples include a method for adding components that are deficient at high temperatures, a wax barrier method, a method using a DNA polymerase antibody or an abutama, and a method using a DNA polymerase that is activated by heat treatment. .
  • these methods have problems such as complicated operations and the need for expensive reagents such as antibodies. In addition, it cannot be used for amplification reactions that originally have a low reaction temperature.
  • an object of the present invention is to provide an efficient and easy-to-operate method for preventing amplification of a non-specific amplification product due to formation of a primer dimer.
  • the present invention provides a novel method for suppressing non-specific amplification due to the formation of the primer dimer as described above.
  • the inventors added a substance that binds to a dimer dimer, such as a mismatch binding protein or a recombinant enzyme protein, to the amplification reaction solution, thereby non-specific due to the formation of the primer dimer. It has been found that amplification can be significantly reduced. These proteins bind to the non-complementary pairing part of the primer dimer and stop the DNA strand extension reaction from the 3 ′ end by the DNA polymerase. As a result, primer dimer From one, non-specific amplification is reduced.
  • a dimer dimer such as a mismatch binding protein or a recombinant enzyme protein
  • the present invention provides a method for suppressing a nonspecific amplification reaction associated with primer dimer formation, which comprises adding a substance that binds to a primer dimer in a nucleic acid amplification reaction to a sample.
  • a substance that binds to the primer dimer a substance having mismatch recognition ability, preferably a mismatch binding protein can be used.
  • the mismatch binding protein include MutS, MSH2, MSH6, or a mixture thereof.
  • the substance that binds to the primer dimer can be added to the sample at a concentration of 0.1% to 10%, preferably 0.5% to 2%, more preferably 0.7% to 1.3%.
  • a substance having a DNA recombination ability preferably a DNA recombination enzyme
  • DNA recombinant enzymes include RecA protein, T4 gene or single-stranded binding protein
  • a nucleic acid of interest comprising a substance that binds to a primer dimer, a step of adding a primer to a sample, and a step of incubating the sample.
  • a method for amplifying a region, a method for determining the presence or absence of a target nucleic acid sequence, or a method for determining the presence or absence of a mutation, deletion and Z or insertion in a target nucleic acid sequence are provided.
  • a substance having mismatch recognition ability preferably a mismatch binding protein can be used as the substance that binds to the primer dimer.
  • the mismatch-binding protein include MutS, MSH2, MSH6, or a mixture thereof.
  • a substance having DNA recombination ability preferably a DNA recombination enzyme
  • a DNA recombination enzyme can be used as the substance that binds to the primer dimer.
  • An example of the DNA recombination enzyme is RecA protein.
  • a PCR method or an isothermal amplification method can be used as a nucleic acid amplification method in these methods.
  • the primer used in the isothermal amplification method includes at least two kinds of primer sets capable of amplifying the target nucleic acid sequence, and the first primer included in the primer set is a target nucleic acid sequence.
  • a sequence ( ⁇ ′) that hybridizes to the complementary sequence (Be) of (B) is included on the 5 ′ side of the sequence (Ac ′), and the second primer included in the primer set is the target nucleic acid.
  • a mismatch binding protein or a recombinant enzyme protein binds to a mismatch sequence that is a force between primers, suppresses the amplification, and further causes only the target sequence-specific amplification, The effect of improving sensitivity and specificity for amplifying the target sequence can be obtained.
  • This method can be applied to a wide range of amplification and detection fields from infectious diseases to SNPs and mutations.
  • FIG. 1 is a diagram showing the positional relationship of each primer region with respect to the human STS DYS237 gene.
  • FIG. 2 is a diagram showing the three-dimensional structure of forward primer F1.
  • FIG. 3 is a graph showing the results of Example 1.
  • FIG. 4 is a diagram showing the positional relationship of each primer with respect to the CYP2C 19 sequence.
  • FIG. 5 shows the results of Example 2.
  • mismatch binding protein (“mismatch recognition protein”).
  • MutS protein bound to MutS protein JP 9-504699
  • MutM protein JP 2000-300265
  • GFP Green Fluore scence Protein
  • mismatch means a set of base pairs selected from adenine ( ⁇ ), guanine (G), cytosine (C), and thymine (T) (uracil (U) in the case of RNA). Is not a normal salt pair (a combination of ⁇ and ⁇ or a combination of G and C). Mismatches include not only one mismatch, but also multiple consecutive mismatches, mismatches caused by insertions and deletions of one or more bases, and combinations thereof.
  • a primer dimer has a double-stranded structure in which a part or the whole of a primer is paired with another primer and includes a non-complementary region.
  • Such a heteroduplex structure results in a false amplification product that should not be produced. Therefore, if a mismatch binding protein is added to the reaction solution used for the nucleic acid amplification reaction, the mismatch binding protein binds to the heteroduplex structure as described above, and the subsequent amplification reaction is hindered. Therefore, by using a mismatch binding protein, it is possible to prevent the generation of an erroneous amplification product.
  • the mismatch binding protein used in the present invention may be any protein known to those skilled in the art as long as it is a protein that recognizes a mismatch in a double-stranded nucleic acid and can bind to the mismatch site. It may be a thing.
  • Mismatch binding proteins used include MutS, MutH, MutL, HexA, MSH1-6, Rep3, RNaseA, uracil DNA glycosidase, T4 endonuclease VII, and resolvase.
  • it is MutS, MSH2 or MSH6, or a mixture of two or more thereof, more preferably a force that is MutS, but is not limited thereto.
  • the mismatch binding protein used in the present invention has one or more amino acid substitutions, deletions, additions, and Z in the amino acid sequence of the wild-type protein as long as the mismatch in the double-stranded nucleic acid can be recognized.
  • it may be an inserted amino acid sequence protein (variant).
  • Such mutants can also be created artificially by forces that may occur in nature.
  • site-directed mutagenesis methods include WP DengiJ. A. Nickoloff's method (An al. Biochem., 200, 81, 1992), KL Makamaye and F. Eckstein's method (Nucleic Adids Res., 14 , 9679-9698, 1986), and random mutagenesis methods include methods using E. coli XL1-Red strain (Stratagene) deficient in the basic repair system, sodium nitrite, etc. CF. — J. Diaz et al., BioTechnique, 11, 204— 211, 1991).
  • mismatch binding proteins such as MutM, MutS and their analogs (Radman, M. et al., Annu. Rev. Genet. 20: 523 -538 ( 1986); Radaman, M. etal., Sci. Amer., August 1988, pp40-46; Modrich, P., J. Biol. Chem. 264: 6597-6600 (1989); Lahue, RS et al., Science245 : 160-164 (1988); Jiricny, J. et al,. Nucl. Acids Res. 16: 7843-7853 (1988); Su, SS et al., J. Biol. Chem.
  • the mismatch binding protein may also bind to a single-stranded nucleic acid, and the binding of such a mismatch binding protein to a single-stranded nucleic acid is inhibited by the single-stranded binding protein. It is known that Therefore, when a mismatch binding protein is used in the mutation detection method according to the present invention, it is preferable to use a single chain binding protein in combination.
  • the single-stranded binding protein (SSB) used to inhibit the binding of the mismatch binding protein to the single-stranded nucleic acid can be any SSB known in the art.
  • Preferred SSBs include single-stranded binding proteins from E. coli, Drosophila, and Xenopus laevis, and the gene 32 protein from T4 butteriophage, and their equivalents from other species. It is not limited to.
  • the single-stranded binding protein used in the present invention has one or more amino acid substitutions, deletions, attached calories and Z in the amino acid sequence of the wild-type protein as long as it can bind to the single-stranded nucleic acid.
  • it may be a protein (mutant) having an inserted amino acid sequence ability. Such mutants can also be created artificially by forces that may occur in nature.
  • the mismatch binding protein may bind to a double-stranded nucleic acid that does not contain a mismatch, and the mismatch binding protein is activated by using an activator. It is known that such mismatch binding protein can be prevented from erroneous binding. Therefore, when a mismatch binding protein is used in the mutation detection method according to the present invention, it is preferable to use a protein that has been activated by a mismatch binding protein activator.
  • An active agent for activating the mismatch binding protein can be appropriately selected by those skilled in the art.
  • active agents for activating mismatch binding proteins ATP (adenosine 5, monotriphosphate), ADP (adenosine 5, monodiphosphate), ATP— ⁇ S (adenosine 5,- 0- (3-thiotriphosphate)), AMP- ⁇ (adenosine 5, mono [ ⁇ , y imido] triphosphate), or one of the nucleotides that can bind to mismatch-binding proteins. It is not limited to these.
  • the activity of the mismatch binding protein can be performed by incubating the mismatch binding protein and the active agent at room temperature for several seconds to several minutes.
  • the recombinant enzyme protein used in the present invention can be appropriately selected by those skilled in the art.
  • human genomic DNA manufactured by Clontech
  • the target nucleic acid sequence in the human STS DYS237 gene contained therein was amplified.
  • a primer a primer pair having the following sequence was used. The positional relationship of each primer region with respect to the saddle type was as shown in FIG. 1 (SEQ ID NO: 1).
  • the sequence on the 3 'end side 22mer: underlined portion
  • the sequence on the 5' end side (16mer: other than the underlined portion
  • Reverse primer R1 has its 3 terminal sequence (20mer: underlined) annealed in a saddle shape, and after extension reaction, 5 'terminal sequence (lOmer: other than underlined) 1S depends on the primer It is designed to hybridize to the region on the extension strand that begins 16 bases downstream of the 3 'terminal residue of the primer.
  • R1 GCAGCATCACCAACCCAAAAGCACTGAGTA (SEQ ID NO: 2)
  • a 25 ⁇ L reaction solution having the following composition was prepared and incubated at 60 ° C for 1 hour.
  • the type ⁇ ⁇ was allowed to react with double strands.
  • the same experiment was performed on a solution to which sterilized water was added instead of the bowl.
  • the composition of the reaction solution is as follows. Tris-HCl (20mM, pH8.8), KCl (10m
  • Outer primer R3Z SEQ ID NO: 4 5,-AGGGTTGTTG ATGTCC ATC 3 '
  • Table 2 shows the composition of each sample reaction solution. [0045] [Table 2]

Abstract

L’invention concerne une méthode de suppression de la réaction d’amplification non-spécifique accompagnant la formation d’une amorce de dimère au cours d’une réaction d’amplification d'acide nucléique qui se caractérise par l’ajout à un échantillon d'une substance capable de se lier à l'amorce de dimère. En tant que substance capable de se lier à l’amorce de dimère, l'utilisation d'une substance capable de reconnaître un mésappariement tel qu'une protéine d’association des appariements ou l’utilisation d’une substance pouvant se recombiner génétiquement comme une recombinaison d’ADN est possible.
PCT/JP2006/305372 2005-03-17 2006-03-17 Methode pour diminuer l’amplification non-specifique d’une amorce de dimere WO2006098428A1 (fr)

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JP2005-076529 2005-03-17
JP2005076529A JP2006254784A (ja) 2005-03-17 2005-03-17 プライマーダイマーからの非特異的増幅を減少させる方法

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008136436A (ja) * 2006-12-04 2008-06-19 Fujifilm Corp １本鎖ｄｎａ結合蛋白質を用いた核酸の変異検出方法
CN111187805A (zh) * 2020-01-19 2020-05-22 陕西师范大学 一种辅助突变引物的设计方法及其应用
CN113832147A (zh) * 2021-09-08 2021-12-24 华南农业大学 一种高效的大片段dna合成与扩增的pcr引物、方法及应用

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KR100987352B1 (ko) * 2008-04-15 2010-10-12 주식회사 인트론바이오테크놀로지 비특이 증폭을 감소시킬 수 있는 ｐｃｒ용 프라이머 및이를 이용한 ｐｃｒ 방법
CA2749693C (fr) 2009-01-15 2018-11-06 Hokkaido Mitsui Chemicals Inc. Preparation enzymatique contenant de l'adn polymerase thermostable, procede de production de ladite preparation et procede de detection de l'organisme devant etre detecte
WO2011046972A2 (fr) * 2009-10-12 2011-04-21 Life Technologies Corporation Compositions et procédés pour supprimer les interactions d'amorces
KR101742681B1 (ko) 2015-01-30 2017-06-01 에스디 바이오센서 주식회사 상보적 염기서열 내지는 미스-매치된 염기를 포함하는 상보적인 염기서열과 연결된 pcr 프라이머 및 이를 이용한 핵산 증폭 방법

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JPH11511010A (ja) * 1995-06-06 1999-09-28 ザ・マウント・シナイ・メデイカル・センター・オブ・ザ・シテイ・ユニバーシテイ・オブ・ニユーヨーク 熱安定性ｍｕｔＳ遺伝子のクローニングおよび発現ならびにその蛋白質および使用法
JP2001510681A (ja) * 1997-07-01 2001-08-07 ローン・パーク・リサーチ・インコーポレーテッド 蛍光強度消光効果を用いる溶液中のヌクレオチドのアッセイ
WO2002024902A1 (fr) * 2000-09-19 2002-03-28 Eiken Kagaku Kabushiki Kaisha Procede permettant de synthetiser un polynucleotide
JP2003259882A (ja) * 2001-12-19 2003-09-16 F Hoffmann La Roche Ag 改良されたｐｃｒ用の試薬

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JPH11511010A (ja) * 1995-06-06 1999-09-28 ザ・マウント・シナイ・メデイカル・センター・オブ・ザ・シテイ・ユニバーシテイ・オブ・ニユーヨーク 熱安定性ｍｕｔＳ遺伝子のクローニングおよび発現ならびにその蛋白質および使用法
JP2001510681A (ja) * 1997-07-01 2001-08-07 ローン・パーク・リサーチ・インコーポレーテッド 蛍光強度消光効果を用いる溶液中のヌクレオチドのアッセイ
WO1999010369A1 (fr) * 1997-08-28 1999-03-04 Thomas Jefferson University Compositions, kits et procedes permettant d'effectuer la modulation par des nucleotides d'adenine de proteines de reconnaissance du defaut de complementarite de l'adn
WO2002024902A1 (fr) * 2000-09-19 2002-03-28 Eiken Kagaku Kabushiki Kaisha Procede permettant de synthetiser un polynucleotide
JP2003259882A (ja) * 2001-12-19 2003-09-16 F Hoffmann La Roche Ag 改良されたｐｃｒ用の試薬

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008136436A (ja) * 2006-12-04 2008-06-19 Fujifilm Corp １本鎖ｄｎａ結合蛋白質を用いた核酸の変異検出方法
CN111187805A (zh) * 2020-01-19 2020-05-22 陕西师范大学 一种辅助突变引物的设计方法及其应用
CN111187805B (zh) * 2020-01-19 2023-06-23 陕西师范大学 一种辅助突变引物的设计方法及其应用
CN113832147A (zh) * 2021-09-08 2021-12-24 华南农业大学 一种高效的大片段dna合成与扩增的pcr引物、方法及应用

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