WO2006051988A1

WO2006051988A1 - Method of nucleic acid amplification

Info

Publication number: WO2006051988A1
Application number: PCT/JP2005/020960
Authority: WO
Inventors: Yoshihide Hayashizaki; Toshizo Hayashi; Yasumasa Mitani
Original assignee: Riken; Kabushiki Kaisha Dnaform
Priority date: 2004-11-15
Filing date: 2005-11-15
Publication date: 2006-05-18

Abstract

A method of nucleic acid amplification exhibiting a high amplification efficiency. There is provided a method of amplifying a double-stranded target nucleic acid containing a target nucleic acid sequence contained in a template nucleic acid with the use of a nucleic acid synthetase and a primer, comprising the step (i) of providing at least one nucleotide molecule capable of interposing between the two strands of the double-stranded target nucleic acid to thereby attain dissociation of a primer binding region of either of the strands into a single stranded form and the step (ii) of preparing a nucleic acid amplification reaction mixture containing the above at least one nucleotide molecule, template nucleic acid, primer and nucleic acid synthetase and carrying out a nucleic acid amplification reaction with the use of the nucleic acid amplification reaction mixture.

Description

Specification

Nucleic acid amplification method

Reference to related applications

[0001] This patent application consists of previously filed US Provisional Application No. 60Z627,182 (filing date: November 15, 2004) and Japanese Patent Application No. 2004-380275 (filing date: 200). This is accompanied by a priority claim based on December 28, 4 years). The entire disclosure of these earlier patent applications is hereby incorporated by reference.

Background of the Invention

[0002] Field of the Invention

The present invention relates to a method for amplifying a target nucleic acid sequence of interest.

[0003] Art of Putuo

In recent years, 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.

[0004] Conventional nucleic acid amplification methods include the most well-known PCR methods (US Pat. No. 4,683,195; US Pat. No. 4,683,202; and US Pat. No. 4,800,159), RNA An RT-PCR method (Trends in Biotechnology 10, ppl4 6-152, 1992) is known. These methods have problems such as requiring complex temperature control and contamination with non-specific amplification products due to primer misannealing.

[0005] As a nucleic acid amplification method, an isothermal amplification method that does not require complicated temperature control as in the PCR method is known. For example, a strand displacement amplification method (SDA method; Japanese Patent Publication No. 7-114718) Publication), self-sustained sequence amplification method (3SR method), Q β replicase method (Japanese Patent No. 2710159 publication), NASBA method (Japanese Patent No. 2650159 publication), LAMP method (International Publication No. ΟθΖ28082 pamphlet) The methods described in the 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. [0006] For quick and accurate examination and diagnosis, it was necessary to solve these problems.

Summary of the Invention

[0007] In the nucleic acid amplification method using a nucleic acid synthase and a primer, 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. By carrying out the 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.

[0008] Accordingly, an object of the present invention is to provide a nucleic acid amplification method having high amplification efficiency.

[0009] The nucleic acid amplification method according to the present invention 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.

[0010] According to the present invention, in the nucleic acid amplification method, 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.

Brief Description of Drawings

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] 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 | region.

[FIG. 4] 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.

Explanation of symbols

[0012] 1 Double-stranded nucleic acid to be cage-shaped

2 Target region on the antisense strand

3 Target region on the sense strand

4 First bridge nucleotide molecule

5 Second bridged nucleotide molecule

6 Forward primer

7 Reverse primer

8 Amplification product with target nucleic acid sequence (antisense)

9 Amplification product with target nucleic acid sequence (sense)

10 Amplification products

Detailed description of the invention

[0013] In the nucleic acid amplification method according to the present invention, 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”) are prepared.

In the present invention, “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.

[0015] 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.

[0016] According to a preferred embodiment of the present invention, 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.

[0017] 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. As a result, 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. As a result of such a reaction being repeated, the main type is an amplification product (8 and 9) having only the sequence of the target region (2 and 3) (FIG. 1 (b)). Next, 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). As a result, 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)).

[0018] In the present invention, "nobly" means that a 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. For example, J. Sam brook, EF Frisch, T. Maniatis; Molecular, 2nd edition, old Spring Harbor Laboratory (1989) can be referred to. For example, 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. According to a preferred V, embodiment of the present invention, the nucleotide moiety that hybridizes to a 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.

[0019] In the bridged 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. Is preferred. Therefore, according to a preferred embodiment of the present invention, 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. According to another preferred embodiment of the present invention, 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 .

[0020] 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,. For example, 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.

[0021] 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. For example, 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. Alternatively, the intervening sequence This sequence is not found in the nucleic acid sequence present in the reaction solution! It may be a / ヽ array.

[0022] It is preferable that the 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.

[0023] Preferably, 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. Such 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.

[0024] According to a particularly preferred embodiment of the present invention, the bridge nucleotide molecule is designed as shown in FIG. First, in the target nucleic acid sequence and its complementary sequence, as shown in FIG. 2 (a), a region used for designing the bridge nucleotide molecule is selected. Here, Fw represents a sequence contained in the first primer, and Fw ′ represents a sequence complementary thereto. Rv represents a sequence contained in the reverse primer, and 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). Here, ten thymine residues (T) are used as examples of intervening sequences! /, But this is not a limitation! /.

[0025] In the nucleic acid amplification method according to the present invention, a 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.

[0026] According to a preferred embodiment of the present invention, the nucleic acid amplification reaction solution contains two or more kinds of bridged nucleotide molecules. In this embodiment, for example, 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.

[0027] 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. As such a primer, 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.

[0028] 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. st”) and Notell's caldotenax (hereinafter referred to as “B. ca”). 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, Φ 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.

[0029] The nucleic acid amplification reaction can be performed under isothermal conditions. Here, “isothermal” refers to maintaining an approximately constant temperature condition such that the enzyme and the primer can substantially function. Furthermore, “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.

[0030] 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. In this nucleic acid amplification reaction, in order for the primer to anneal to the target nucleic acid, for example, 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.

[0031] Other 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. In addition, additives such as dimethyl sulfoxide and betaine (Ν, Ν, Ν-trimethylglycine), acidic substances described in WO 99/54455 pamphlets, cation complexes, etc. may be used. Yo! /

[0032] In order to carry out the nucleic acid amplification method according to the present invention, necessary reagents and the like can be combined into a kit. Therefore, according to the present invention, there is provided a kit 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.

[0033] According to a preferred embodiment of the present invention, 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.

[0034] 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

[0035] The present invention will be specifically described below with reference to examples, but the scope of the present invention is not limited to these examples.

[0036] Example 1: Amplification of target nucleic acid sequence in Ascl3 gene using bridged nucleotide molecule In this example, a bridged nucleotide molecule was used to amplify the target nucleic acid sequence in the Ascl3 gene by a reaction under isothermal conditions.

[0037] (1) Preparation of plasmid used as cage

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.

[0038] (2) Preparation of bridged nucleotide molecules and primers

In the Ascl3 gene partial sequence (SEQ ID NO: 1) to be amplified, each region to be used for designing the bridged nucleotide molecule and the primer was determined as shown in FIG. In FIG. 3, Al, A2, CI, and C2 represent regions used for designing the bridge nucleotide molecule, and Fw and Rv represent the forward primer and the reverse primer, respectively.

[0039] As a bridged nucleotide molecule, 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.

[0040] Oligo set 1:

Oligo 1-1: 5 tggagggctg tttttttttt gacaggctct-3 '(SEQ ID NO: 2);

Oligo 1-2: 5 and tccctggacc tttttttttt gccgcagctg-3 '(SEQ ID NO: 3).

[0041] Oligo set 2:

Oligo 2-1: 5'- gacaggctct tttttttttt tggagggctg-3 '(SEQ ID NO: 4);

Oligo 2-2: 5 '-gccgcagctg tttttttttt tccctggacc- 3' (酉自 5 [J number 5).

[0042] As primers, oligonucleotides having the following sequences were synthesized:

Fw: 5 -ATGGACACCAGAAGCTACCC-3 '(SEQ ID NO: 6);

Rv: 5 -TCAAATGACTCTCAGAGCCG-3 '(SEQ ID NO: 7).

[0043] (3) Amplification reaction Reaction solution (25 μL) 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%

4 2 4 4

), DNTP (l. 4 mM), 2000 nM each of the above primer pairs, each 2000η are both), and a truncated plasmid (lng), and 16 U Bst DNA polymerase (NEW ENGLAND BioLabs). Was incubated at 60 ° C for 1 hour. As a control, the same experiment was performed on a reaction solution containing no bridged nucleotide molecule.

[0044] (4) Electrophoresis

For 5 μ1 of each reaction solution after amplification reaction, 3% NuSieve 3: 1 Agarose (manufactured by BioWhittaker Molecular Applications (BMA); purchased from Takara Bio Inc .; “NuSieve” is a registered trademark of BMA) And electrophoresed at 100V for 80 minutes. Nucleic acids were detected by staining the gel after electrophoresis with ethidium bromide (EtBr). The results are shown in Fig. 5. Samples in each lane in Fig. 5 are as follows: Lane 1: Size marker (pHY marker); Lane 2: No bridge nucleotide molecule included! /, Reaction solution; Lane 3: Oligoset 1 as a bridge nucleotide molecule Lane 4: reaction solution containing oligo set 2 as bridge nucleotide molecule; lane 5: reaction solution containing both oligo set 1 and oligo set 2 as bridge nucleotide molecule.

[0045] According to FIG. 5, 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. These results reveal that the amount of amplification of the target nucleic acid sequence is remarkably increased by adding bridge nucleotide molecules to the nucleic acid amplification reaction system.

Claims

The scope of the claims

[1] A method 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,

(i) a step of preparing at least one nucleotide molecule capable of dissociating a primer binding region in either strand into a single strand state by intervening between both strands of the double-stranded target nucleic acid; And

(ii) A method comprising a step of preparing a nucleic acid amplification reaction solution containing at least one kind of the above-mentioned nucleotide molecules, a truncated nucleic acid, a primer and a nucleic acid synthesis enzyme, and performing a nucleic acid amplification reaction using the solution.

[2] The nucleotide molecule has a first binding sequence that hybridizes to the first strand of the double-stranded target nucleic acid at one end and a second binding sequence that hybridizes to the second strand. The method according to claim 1, which is at the other end.

[3] 3 ′ terminal residue of the region on the first strand where the first binding sequence is hybridized 20 nucleotides downstream from the 5 ′ terminal residue of the primer binding region on the first strand 3. The method according to claim 2, wherein the method is located 100 nucleotides upstream.

[4] 3 'terminal residue of the region on the second strand where the second binding sequence is hybridized 20 nucleotides downstream from the 5' terminal residue of the primer binding region on the second strand 3. The method according to claim 2, wherein the method is located 100 nucleotides upstream.

[5] The method according to claim 2, wherein the first binding sequence and the second binding sequence contained in the nucleotide molecule are not complementary to each other.

[6] The method according to claim 1, wherein the nucleic acid amplification reaction solution contains two or more kinds of the nucleotide molecules.

[7] The method according to claim 1, wherein the nucleic acid synthase has a strand displacement activity.

[8] The method according to [1], wherein the nucleic acid amplification reaction is performed isothermally.

[9] A kit 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, between both strands of the double-stranded target nucleic acid A kit comprising at least one nucleotide molecule capable of dissociating a primer binding region in either strand into a single strand by intervening.

[10] The nucleotide molecule has a first binding sequence that hybridizes to the first strand of the double-stranded target nucleic acid at one end, and a second binding sequence that hybridizes to the second strand. 10. The kit according to claim 9, which is at the other end.

[11] 3 ′ terminal residue of the region on the first strand to which the first binding sequence hybridizes 20 nucleotides downstream to 5 ′ terminal residue of the primer binding region on the first strand to 100 The kit according to claim 10, which is located upstream of the nucleotide.

[12] The 3 ′ terminal residue of the region on the second strand where the second binding sequence is hybridized 20 nucleotides downstream from the 5 ′ terminal residue of the primer binding region on the second strand 11. The kit according to claim 10, which is located 100 nucleotides upstream.

[13] The kit according to claim 10, wherein the first binding sequence and the second binding sequence contained in the nucleotide molecule are not complementary to each other.

[14] The kit according to claim 9, comprising two or more kinds of the nucleotide molecules.

[15] The kit according to claim 9, further comprising a nucleic acid synthase having strand displacement activity.