WO2006051988A1 - Procédé d'amplification d'acide nucléique - Google Patents

Procédé d'amplification d'acide nucléique Download PDF

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Publication number
WO2006051988A1
WO2006051988A1 PCT/JP2005/020960 JP2005020960W WO2006051988A1 WO 2006051988 A1 WO2006051988 A1 WO 2006051988A1 JP 2005020960 W JP2005020960 W JP 2005020960W WO 2006051988 A1 WO2006051988 A1 WO 2006051988A1
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WO
WIPO (PCT)
Prior art keywords
nucleic acid
strand
primer
nucleotide molecule
sequence
Prior art date
Application number
PCT/JP2005/020960
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English (en)
Japanese (ja)
Inventor
Yoshihide Hayashizaki
Toshizo Hayashi
Yasumasa Mitani
Original Assignee
Riken
Kabushiki Kaisha Dnaform
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from JP2004380275A external-priority patent/JP2007330101A/ja
Application filed by Riken, Kabushiki Kaisha Dnaform filed Critical Riken
Publication of WO2006051988A1 publication Critical patent/WO2006051988A1/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

Definitions

  • the present invention relates to a method for amplifying a target nucleic acid sequence of interest.
  • nucleic acid detection methods have been widely used in genetic diagnosis, nucleic acid testing of agricultural products, infectious disease diagnosis, and the like, and nucleic acid amplification technology is the basic technology for these.
  • a nucleic acid amplification method As a nucleic acid amplification method, an isothermal amplification method that does not require complicated temperature control as in the PCR method is known.
  • a strand displacement amplification method SDA method; Japanese Patent Publication No. 7-114718) Publication
  • self-sustained sequence amplification method (3SR method)
  • Q ⁇ replicase method Japanese Patent No. 2710159 publication
  • NASBA method Japanese Patent No. 2650159 publication
  • LAMP method International Publication No. ⁇ 28082 pamphlet
  • ICAN method WO02Z16639 pamphlet
  • the rolling circle method and the WO2004Z040019 pamphlet are known. These methods still have some points to be improved, such as complicated reaction, difficult primer design, and low amplification efficiency.
  • the present inventors have added a nucleotide molecule intervening between both strands of a double-stranded target nucleic acid to be amplified to a reaction solution to add a nucleic acid.
  • amplification reaction it was found that the primer binding region in the double-stranded target nucleic acid can be dissociated into a single-stranded state, thereby significantly improving the amplification efficiency of the nucleic acid amplification reaction.
  • the present invention is based on these findings.
  • an object of the present invention is to provide a nucleic acid amplification method having high amplification efficiency.
  • the nucleic acid amplification method is a method for amplifying a double-stranded target nucleic acid containing a target nucleic acid sequence contained in a truncated nucleic acid using a nucleic acid synthetase and a primer, and comprising the steps of (i) Providing at least one nucleotide molecule capable of dissociating the primer-binding region of either strand into a single strand by intervening between both strands of the double-stranded target nucleic acid; and (ii ) Comprising a step of preparing a nucleic acid amplification reaction solution containing at least one kind of the aforementioned nucleotide molecule, cage nucleic acid, primer and nucleic acid synthase, and performing a nucleic acid amplification reaction using the solution.
  • the amplification efficiency can be improved, and misannealing of a primer to a non-target region can be reduced. Furthermore, since the nucleic acid amplification method according to the present invention can be carried out under isothermal conditions, complicated temperature control is not required.
  • FIG. 1 is a diagram schematically showing a possible mechanism of action for a nucleic acid amplification method according to the present invention.
  • FIG. 2 is a diagram showing the structure of a bridged nucleotide molecule according to a preferred embodiment.
  • FIG. 3 shows a partial sequence (SEQ ID NO: 1) of Ascl3 gene targeted for amplification in the Examples, and each region used for designing bridge nucleotide molecules and primers. It is a figure which shows the position of an area
  • FIG. 4 is a diagram showing the nucleotide sequences of the bridged nucleotide molecules used in the Examples and their positions on the target nucleic acid sequence to be hybridized.
  • FIG. 5 is an electrophoretogram showing the results of the amplification reaction in the examples.
  • nucleic acid amplification method first, by intervening between both strands of a double-stranded target nucleic acid, the primer-binding region in any strand can be dissociated into a single-stranded state.
  • Nucleotide molecules hereinafter “bridge nucleotide molecules”.
  • nucleotide molecule is used in the meaning including DNA, RNA, and PNA (peptide nucleic acid add). According to a preferred embodiment of the invention, the nucleotide molecule is DNA.
  • the bridge nucleotide molecule can intervene between the two strands in a partial region of the double-stranded target nucleic acid and unravel the double-stranded structure of that portion without destroying the nucleic acid molecule itself. To do. By such intervention of the bridge nucleotide molecule, the double-stranded structure in the peripheral part of the region is unwound and partially becomes a single-stranded state. Bridged nucleotide molecules contain a primer binding region in the single-stranded part in this way. Designed to be rare.
  • the bridged nucleotide molecule has a first binding sequence that hybridizes to the first strand of the double-stranded target nucleic acid at one end, and the second It has a second binding sequence that hybridizes to the strand at the other end.
  • Figure 1 shows the mechanism of action of these two types of bridged nucleotide molecules.
  • the first bridging nucleotide molecule (4) and the second bridging nucleotide molecule (5) present in the reaction solution enter the target region in the double-stranded nucleic acid (1), which is a cage, A structure as shown in 1 (a) is formed.
  • the forward primer (6) and the reverse primer (7) hybridize to the primer binding region in a single-stranded state, and then a primer extension reaction by a nucleic acid synthase occurs.
  • the main type is an amplification product (8 and 9) having only the sequence of the target region (2 and 3) (FIG. 1 (b)).
  • the pride nucleotide molecules (4 and 5) also act on these amplification products in the form of cages to form a structure as shown in FIG. 1 (c).
  • the forward primer (6) and the reverse primer (7) hybridize to the primer binding region in a single-stranded state, and then a primer extension reaction by a nucleic acid synthase occurs. By repeating such a reaction, a large amount of amplification product (10) is efficiently formed (Fig. L (d)).
  • nucleotide molecule hybridizes to a target nucleotide molecule under stringent conditions and does not hybridize to nucleotide molecules other than the target nucleotide molecule.
  • Stringent conditions can be determined depending on the melting temperature Tm (° C) of the duplex of the specific nucleotide molecule and its complementary strand, the salt concentration of the hybridization solution, etc.
  • Tm melting temperature
  • the nucleotide molecule when hybridization is performed at a temperature slightly lower than the melting temperature of the nucleic acid molecule to be used, the nucleotide molecule can be specifically hybridized to the target nucleotide molecule.
  • a child is said to comprise a sequence of all or part of a nucleotide molecule complementary to its target nucleotide molecule.
  • the first binding sequence or the second binding sequence hybridizes to a region adjacent to the primer binding region in the first strand or the second strand of the double-stranded target nucleic acid.
  • the bridge nucleotide molecule is such that the 3 ′ terminal residue of the region on the first strand where the first binding sequence is hybridized is on the first strand. Designed to be located 20 nucleotides downstream to 100 nucleotides upstream, more preferably 10 nucleotides downstream to 50 nucleotides upstream, more preferably 1 to 20 nucleotides upstream from the 5 'terminal residue of the primer binding region. Is done.
  • the bridge nucleotide molecule is such that the 3 ′ terminal residue of the region on the second strand to which the second binding sequence hybridizes is on the second strand. Designed to be located 20 nucleotides downstream to 100 nucleotides upstream, more preferably 10 nucleotides downstream to 50 nucleotides upstream, even more preferably 1 to 20 nucleotides upstream with respect to the 5 'terminal residue of the primer binding region .
  • the chain lengths of the first binding sequence and the second binding sequence are particularly limited as long as the binding specificity is not impaired between the nucleic acid sequences present in the reaction solution. Nah,.
  • the chain lengths of the first binding sequence and the second binding sequence are each independently 5 to 100 nucleotides, preferably 5 to 50 nucleotides, more preferably 5 to 30 nucleotides, and even more preferably 7 It can be -20 nucleotides, more preferably 8-15 nucleotides.
  • the bridge nucleotide molecule may include an intervening sequence between the first binding sequence and the second binding sequence.
  • the chain length of such intervening sequences is 1 or more nucleotides, preferably 3 to: L00 nucleotides, more preferably 5 to 50 nucleotides, and even more preferably 8 to 30 nucleotides.
  • the specific nucleotide sequence of the intervening sequence is not particularly limited as long as it is a sequence that does not affect the mechanism of action of the bridge nucleotide molecule as shown in FIG.
  • the nucleotide sequence of the intervening sequence can be a sequence that is not found in either the target nucleic acid sequence or its complement.
  • the intervening sequence This sequence is not found in the nucleic acid sequence present in the reaction solution! It may be a / ⁇ array.
  • first binding sequence and the second binding sequence are not complementary to each other. As a result, bridging nucleotide molecules in a single molecule are avoided. When using multiple types of bridged nucleotide molecules, it is preferable to design each bridged nucleotide molecule so that hybridization between these molecules does not occur.
  • the bridged nucleotide molecule is modified so that an extension reaction by a nucleic acid synthase from the 3 'end does not occur after hybridization to the target nucleic acid sequence.
  • modification may be performed by any method known to those skilled in the art.
  • An example of such a method is a method in which the 3 ′ end of the nucleotide molecule is amination modified.
  • the bridge nucleotide molecule is designed as shown in FIG.
  • a region used for designing the bridge nucleotide molecule is selected.
  • Fw represents a sequence contained in the first primer
  • Fw ′ represents a sequence complementary thereto.
  • Rv represents a sequence contained in the reverse primer
  • Rv ′ represents a sequence complementary thereto.
  • A1, A2, Al, A2, Cl, C2, CI, and C2 indicate the regions used in the design of bridged nucleotide molecules.
  • the sequence of these regions is then used to design bridged nucleotide molecules as shown in FIG. 2 (b).
  • ten thymine residues (T) are used as examples of intervening sequences! /, But this is not a limitation! /.
  • nucleic acid amplification reaction solution containing at least one kind of bridged nucleotide molecule, cage nucleic acid, primer and nucleic acid synthase is prepared, and a nucleic acid amplification reaction using this solution is performed.
  • the nucleic acid amplification reaction solution contains two or more kinds of bridged nucleotide molecules.
  • one bridge nucleotide molecule is designed so that the primer binding region on the first strand of the double-stranded target nucleic acid can be dissociated into a single-stranded state, and another bridge nucleotide is obtained.
  • Molecule, double-stranded target It is possible to design the primer binding region on the second strand of the nucleic acid so that it can be dissociated into a single strand.
  • the primer contained in the nucleic acid amplification reaction solution may be any primer that can amplify the target nucleic acid sequence.
  • Such primers are appropriately designed by those skilled in the art.
  • a primer set comprising a primer that hybridizes to the 3 ′ end portion of the target nucleic acid sequence and a primer that hybridizes to the 3 ′ end portion of the complementary sequence of the target nucleic acid sequence is typically used. be able to.
  • the nucleic acid synthase (polymerase) contained in the nucleic acid amplification reaction solution may be any one having normal temperature, medium temperature, or heat resistance, as long as it is generally used in nucleic acid amplification reactions. Possible force Preferably, it has strand displacement activity (strand displacement ability). In addition, this polymerase may be either a natural body or a mutant with artificial mutations. Examples of such a polymerase include DNA polymerase. Furthermore, it is preferable that this DNA polymerase has substantially no 5 ′ ⁇ 3 ′ exonuclease activity. Such DNA polymerases are thermophilic, such as Bacillus stearothermophilus (hereinafter referred to as “B.
  • B. ca Notell's caldotenax
  • Examples include 5 ′ ⁇ 3 of DNA polymerase derived from Bacillus bacteria, mutants lacking exonuclease activity, and Tarenow fragment of DNA polymerase I derived from E. coli.
  • the DNA polymerases used in the nucleic acid amplification reaction include Vent DNA polymerase, Vent (Exo-) DNA polymerase, DeepVent DNA polymerase, DeepVent (Exo-) DNA polymerase, ⁇ 29 phage DNA polymerase, MS-2 phage Examples include DNA polymerase, Z-Taq DNA polymerase, Pfo DNA polymerase, Pfo turbo DNA polymerase, KOD DNA polymerase, 9 ° Nm DNA polymerase, and Therminater DNA polymerase.
  • the nucleic acid amplification reaction can be performed under isothermal conditions.
  • isothermal refers to maintaining an approximately constant temperature condition such that the enzyme and the primer can substantially function.
  • substantially constant temperature conditions means not only maintaining the set temperature accurately, but also not impairing the substantial functions of the enzyme and the primer! Means allowed.
  • the nucleic acid amplification reaction under a certain temperature condition can be carried out by keeping the temperature at which the activity of the enzyme used can be maintained.
  • the reaction temperature is preferably set to a temperature close to or below the melting temperature (Tm) of the primer. It is preferable to set the stringency level in consideration of the melting temperature (Tm) of the primer. Therefore, this temperature is preferably about 20 ° C to about 75 ° C, more preferably about 35 ° C to about 65 ° C.
  • reagents used in the nucleic acid amplification reaction include, for example, catalysts such as magnesium chloride, magnesium acetate, and magnesium sulfate, substrates such as dNTP mix, tris-hydrochloric acid noffer, tricine buffer, sodium phosphate buffer, phosphorus A buffer such as potassium acid buffer can be used.
  • catalysts such as magnesium chloride, magnesium acetate, and magnesium sulfate
  • substrates such as dNTP mix
  • tris-hydrochloric acid noffer such as tris-hydrochloric acid noffer
  • tricine buffer such as sodium phosphate buffer
  • phosphorus A buffer such as potassium acid buffer
  • additives such as dimethyl sulfoxide and betaine ( ⁇ , ⁇ , ⁇ -trimethylglycine), acidic substances described in WO 99/54455 pamphlets, cation complexes, etc. may be used.
  • kits for amplifying a double-stranded target nucleic acid containing a target nucleic acid sequence contained in a vertical nucleic acid using a nucleic acid synthase and a primer and the kit includes at least one kind. Of the bridged nucleotide molecule.
  • the kit according to the present invention comprises two or more bridged nucleotide molecules as described above. According to another preferred embodiment of the present invention, the kit according to the present invention further comprises the above-mentioned nucleic acid synthase having strand displacement activity.
  • the kit according to the present invention may further contain the above-mentioned reagents such as dNTP and buffer, reaction containers, instructions, and the like.
  • Example 1 Amplification of target nucleic acid sequence in Ascl3 gene using bridged nucleotide molecule
  • a bridged nucleotide molecule was used to amplify the target nucleic acid sequence in the Ascl3 gene by a reaction under isothermal conditions.
  • a plasmid was prepared for use as a cage in the amplification reaction. Specifically, a pUC19 vector into which a partial sequence (SEQ ID NO: 1) of the Ascl3 gene (Mus musculus achaete—scute complex nomolog—like 3 (Drosophila); E. coli was transformed.
  • SEQ ID NO: 1 a partial sequence of the Ascl3 gene (Mus musculus achaete—scute complex nomolog—like 3 (Drosophila); E. coli was transformed.
  • each region to be used for designing the bridged nucleotide molecule and the primer was determined as shown in FIG.
  • Al, A2, CI, and C2 represent regions used for designing the bridge nucleotide molecule
  • Fw and Rv represent the forward primer and the reverse primer, respectively.
  • an oligonucleotide having the following sequence was synthesized. The relationship between these sequences and each region shown in FIG. 3 is shown in FIG. In FIG. 4, A2 ′ and C1 ′ represent complementary sequences of A2 and C1, respectively. In addition, each 3 ′ end was aminated so that no extension reaction from each oligonucleotide occurred during the amplification reaction.
  • Oligo 1-1 5 tggagggctg ttttttttttt gacaggctct-3 '(SEQ ID NO: 2);
  • Oligo 1-2 5 and tccctggacc ttttttttttttt gccgcagctg-3 '(SEQ ID NO: 3).
  • Oligo 2-1 5'- gacaggctct ttttttttttttttt tggagggctg-3 '(SEQ ID NO: 4);
  • Oligo 2-2 5 '-gccgcagctg tttttttttttt tccctggacc- 3' ( ⁇ ⁇ 5 [J number 5).
  • oligonucleotides having the following sequences were synthesized:
  • Amplification reaction Reaction solution (25 ⁇ L) with the following composition: Tris—HCl (20 mM, pH 8.8), KCl (10 mM), (NH 2) SO (10 mM) M MgSO (8 mM) M DMSO (3%), Triton X — 100 (1%
  • the bridge nucleotide molecule is not included! /, A small band (about 500 bp) corresponding to the target amplification product is observed in the sample, and the target nucleic acid sequence is slightly amplified. You can see that In contrast, the sample containing the bridged nucleotide molecule showed a remarkably large band corresponding to the target amplification product.

Abstract

Procédé d'amplification d'acide nucléique présentant une efficacité d'amplification élevée. L'invention concerne un procédé consistant à amplifier un acide nucléique double brin cible contenant une séquence d'acides nucléiques cible contenue dans un acide nucléique matrice en utilisant une acide nucléique synthétase et une amorce, comprenant l'étape (i) consistant à fournir au moins une molécule de nucléotide capable de s'intercaler entre les deux brins de l'acide nucléique double brin cible pour de cette manière réaliser la dissociation d'une région liant l'amorce de l'un ou l'autre des deux brins en une forme simple brin et l'étape (ii) consistant à préparer un mélange de réaction d'amplification d'acide nucléique contenant l'au moins une molécule de nucléotide, l'acide nucléique matrice, l'amorce et l'acide nucléique synthétase ci-dessus et à effectuer une réaction d'amplification d'acide nucléique en utilisant le mélange de réaction d'amplification d'acide nucléique.
PCT/JP2005/020960 2004-11-15 2005-11-15 Procédé d'amplification d'acide nucléique WO2006051988A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US62718204P 2004-11-15 2004-11-15
US60/627,182 2004-11-15
JP2004-380275 2004-12-28
JP2004380275A JP2007330101A (ja) 2004-12-28 2004-12-28 核酸の増幅方法

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011522559A (ja) * 2008-06-11 2011-08-04 ジェネフォーム、テクノロジーズ、リミテッド 等温核酸増幅
WO2018038232A1 (fr) * 2016-08-24 2018-03-01 国立大学法人東北大学 Procédé de production d'un produit d'amplification d'un acide nucléique cible et utilisation dudit procédé
US10227660B2 (en) 2013-04-25 2019-03-12 Orion Diagnostica Oy Strand-invasion based DNA amplification method
US11180787B2 (en) 2014-06-05 2021-11-23 Aidian Oy Strand-invasion based DNA amplification method
CN114045330A (zh) * 2021-12-23 2022-02-15 川北医学院附属医院 一种基于滑动复制的核酸等温扩增方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06510201A (ja) * 1990-05-07 1994-11-17 ダイキン工業株式会社 ダブルｄループ形成の診断応用
JPH07503370A (ja) * 1992-01-23 1995-04-13 サイエンティフィック　ジェネリクス　リミテッド 核酸含有溶液の電気化学的処理による変性方法、および、かかる変性方法を利用する増幅方法、複製方法、検出方法およびキット
JPH07289298A (ja) * 1994-04-18 1995-11-07 Becton Dickinson & Co 好熱酵素を用いる鎖置換増幅法
WO2002090538A1 (fr) * 2001-05-08 2002-11-14 Eiken Kagaku Kabushiki Kaisha Procede de synthese d'acide nucleique

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06510201A (ja) * 1990-05-07 1994-11-17 ダイキン工業株式会社 ダブルｄループ形成の診断応用
JPH07503370A (ja) * 1992-01-23 1995-04-13 サイエンティフィック　ジェネリクス　リミテッド 核酸含有溶液の電気化学的処理による変性方法、および、かかる変性方法を利用する増幅方法、複製方法、検出方法およびキット
JPH07289298A (ja) * 1994-04-18 1995-11-07 Becton Dickinson & Co 好熱酵素を用いる鎖置換増幅法
WO2002090538A1 (fr) * 2001-05-08 2002-11-14 Eiken Kagaku Kabushiki Kaisha Procede de synthese d'acide nucleique

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011522559A (ja) * 2008-06-11 2011-08-04 ジェネフォーム、テクノロジーズ、リミテッド 等温核酸増幅
US9062344B2 (en) 2008-06-11 2015-06-23 Geneform Technologies Limited Isothermal nucleic acid amplification
US9657340B2 (en) 2008-06-11 2017-05-23 Orion Pharma (Uk) Limited Isothermal nucleic acid amplification
EP2660336B1 (fr) * 2008-06-11 2017-08-16 Orion Pharma (UK) Limited Amplification isotherme d'acides nucléiques
US10472659B2 (en) 2008-06-11 2019-11-12 Geneform Technologies Limited Isothermal nucleic acid amplification
US10227660B2 (en) 2013-04-25 2019-03-12 Orion Diagnostica Oy Strand-invasion based DNA amplification method
US11180787B2 (en) 2014-06-05 2021-11-23 Aidian Oy Strand-invasion based DNA amplification method
WO2018038232A1 (fr) * 2016-08-24 2018-03-01 国立大学法人東北大学 Procédé de production d'un produit d'amplification d'un acide nucléique cible et utilisation dudit procédé
CN114045330A (zh) * 2021-12-23 2022-02-15 川北医学院附属医院 一种基于滑动复制的核酸等温扩增方法
CN114045330B (zh) * 2021-12-23 2024-02-09 川北医学院附属医院 一种基于滑动复制的核酸等温扩增方法

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