WO2004046383A1 - Dna amplification method - Google Patents

Dna amplification method Download PDF

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Publication number
WO2004046383A1
WO2004046383A1 PCT/JP2003/014575 JP0314575W WO2004046383A1 WO 2004046383 A1 WO2004046383 A1 WO 2004046383A1 JP 0314575 W JP0314575 W JP 0314575W WO 2004046383 A1 WO2004046383 A1 WO 2004046383A1
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dna
enzyme
dn
amplification
double
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PCT/JP2003/014575
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French (fr)
Japanese (ja)
Inventor
Takao Ando
Fuminori Hoshino
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Bml, Inc.
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Priority to JP2002332017 priority
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Publication of WO2004046383A1 publication Critical patent/WO2004046383A1/en

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844Nucleic acid amplification reactions

Abstract

A DNA amplification method wherein a DNA is amplified by repeating in a liquid phase the following amplification step with the use of the following enzymes (A) and (B): (A) an enzyme having a nicking activity; and (B) a DNA polymerase I, etc. showing no 5’→ 3’ exonuclease activity. Namely, the amplification step comprises: treating a double-stranded DNA to be amplified to thereby newly form (1) a double-stranded DNA having one of the DNA strands of the above-described double-stranded DNA with a new DNA strand and (2) a double-stranded DNA consisting of the two remaining DNA strands, using these two double-stranded DNA’s as the new subject DNA’s to be amplified, and then continuously performing the DNA amplification reaction using the above-described enzymes (A) and (B). According to this method, a gene DNA can be conveniently and efficiently amplified.

Description

Specification

DNA amplification method TECHNICAL FIELD

The present invention is a gene, in particular, an invention relating to the amplification method of the DN A. BACKGROUND

The analysis of gene, it is a matter of course it is necessary to DN A qualitatively good condition, at the same time, it is also very important to ensure a sufficient amount of DNA for analysis. For example, (1) from a small amount of cells, to amplify the gene DN A, and when analyzing mutations such multiple gene regions, (2) culture difficult, only a small amount in the sample of non-existent bacteria, a number of and when performing analysis of DNA, (3) in the case analyzes the like of small amounts of consisting unknown nucleotide sequence DN a, the entire gene region of DN a comprising a sample, a need for a technique to amplify the evenly It made often.

This technique, the entire gene amplification: called (whole genome amplification WG A), to date, several methods have been provided.

Examples, PEP (Primer Extension Preamplif ication ¾ (Zhang, L. et al., Proc. Natl. Acad. Sci. USA., Vol.89, pp. 847-5851, 1992), DO P- PCR (Degenerate Oligonucleotide Primed PCR) method (Cheung, VG and SF

Nelson, Proc. Natl. Acad. Sci. USA., Vol. 93, pp.14676-14679, 1996), MDA (Multiple Displacement Amplification) method (Dean, FB et al., Proc.

Natl. Acad. Sci. USA., Vol.99, pp5261-5266, 2002), and the like are known.

Among the above-mentioned prior art, '

(1) PEP method is a method applying the PCR method, a DN A amplification method using a mixture of random nucleotide sequence of 1 5 bases about the length of the oligonucleotide as a primer. That, PEP method, the primer, for example, to coexist with human genomic DNA or the like to be amplified under appropriate conditions, after the nucleotide sequence was Aniru primer to nucleotide sequence positions matching gene DNA, 3 0 in 4 0 ° C approximately temperature by T aq DNA polymerase, performed DNA extension reaction from the primer (DN a synthesis reaction), first, to amplify the entire gene DN a, further, obtained by the amplification reaction the amplification product as a target that is, performs normal P CR reaction using sequence-specific primers, a method of amplifying the gene of interest DNA.

(2) PO PP CR method, the 5 'side and 3' side of the oligonucleotide having a specific nucleotide sequence, in the center, performed have use primers random nucleotide sequence having a length of about 6 bases, it is a DN a amplification. That is, in the PO PP CR method according More P CR method, PCR cycles beginning force to amplify the target DNA is, at low temperature, perform DNA elongation reaction between time long, overlapping PCR cycles, DNA extension reaction raising the temperature, moreover, by shortening the elongation time, Ru method der performing DNA elongation reaction.

Described above, (l) PEP method also, (2) PO P- PCR method also, the amplification of non-specific DNA occurs, it genes DN A is not uniformly amplified, Moreover, most of the amplified DN A there is less than 1Kb, it is said that there may not be suitable for subsequent genetic analysis.

(3) MDA method is a DNA amplification method using random 6-base sequence of approximately oligonucleotides as primers scratch. That, MDA method is Pakuteri Ofaji of B. subtilis, the DN A polymerase Ichize from excellent DN A strand displacement activity (strand displacement activity) are known to have a phi 2 9 phage, heat-denatured and the DN a to be amplified and the above random primers, 30 ° C approximately in to act on the complex obtained by Ani le, to carry out the elongation reaction of the DNA strands at the same temperature (2 9 optimal phage-derived DNA polymerase the temperature), a way of performing amplification of DNA.

In this MD A method, it is necessary to set the amplification temperature relatively low, in order to perform the desired DN A amplification, Ru observed it takes a long time of about 1 8 hours.

An object to be solved by the present invention is, in view of the problems of these prior art, further, easily and efficiently, Ru near to provide a means for amplifying the gene DNA. Disclosed the inventor's invention is directed to solution Decisive for this problem, the intensive studies. As a result, of the endonuclease, the double-stranded DNA, by utilizing what activity that can nick can be seen, and conceived that it is possible to perform the amplification of the desired gene DN A. That is, the present inventors have the two present Zhen DNA, and an enzyme having an activity that can Rukoto put two click, by using a combination of DNA polymerase zero involved in DNA repair, very conveniently, and, efficient, it found that it is possible to provide full gene amplification means, and completed the present invention.

That is, the present invention provides two enzymes and (A) (B):

(A) a double-stranded DNA, activity (hereinafter, this activity, also called nicking activity), which can be nick enzymes with;

Against double-stranded DNA (B) Nick enters, not nicked, as 鍰型 either the DN A chain, from the position of the nick, by replacing the side of the DNA strand nicked, to form a new DNA strand complementary base sequence for this 錄型 DN a chain, it has activity to form a double-stranded DNA of this 錶型 DNA strand and the new DNA strands, and , exist as single-stranded DNA is replaced with a new DNA strand of the degradation of DN a strand of the nicked side does not perform the enzyme;

Was used, the amplification step described below, by performing repeatedly in the liquid phase, to amplify the DNA, DNA amplification (hereinafter, also referred to as the DN A amplification method) is an invention to provide a. Amplification step: by acting against subject to double-stranded DN A amplification, the double-stranded DNA with (1) either DNA strand and a new DNA strand of the double-stranded DNA, (2 ) double stranded DNA formed by single-stranded DN a mutually replaced, the newly to form, these double-stranded DNA, as a new amplified DNA, the enzyme and (a) ( step of performing a DNA amplification reaction, successively by B). BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is according to the DNA amplification method is a schematic diagram showing the DNA amplification process.

Figure 2 is a photograph showing the electrophoresis image about the partially purified product of endonuclease V Thermotoga maritima.

Figure 3 shows the result of investigation for a random nicking activity of E endonuclease V obtained is a photograph showing the Agarosugeru electrophoresis images.

3

Replacement sheets (Rule 26) Fig. 4, the genomic DNA of Therraus therraophi lus, showing the amplification reaction according to the DN A amplification method is a photograph showing the Agarosugeru electrophoresis images.

Figure 5 is the genomic DNA of H e L a cell, the amount of drawing showing the temporal change of the amplification reaction producing compounds according to the DNA 增幅 method.

Figure 6 was used Ve nt (exo single DNA polymerase, indicating the amplification reaction product according to the DNA amplification method is a photograph showing the Agarosugeru electrophoresis images.

Figure 7 used the E endonuclease N. B st NBI having a specific nicking activity shows 增幅 reaction products according to the DNA amplification method is a photograph showing the Agarosugeru electrophoresis images. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, describing aspects of the present invention.

This DN A amplification method, an enzyme (A) having a nicking activity, is a DNA amplification method using an enzyme (B) having the above polymerase. One peptidase activity.

1. For the enzyme (A)

As mentioned above, the enzyme (A) is an enzyme that has nicking activity, in the secondary Kkingu activity, and activity that can contain two click anywhere in the double-stranded DN A, two activity that can nick at a specific position of the chain DNA, include both.

(1) Examples of the enzymes (A), first, an arbitrary position of the double-stranded DNA, the activity capable of nicking with a (hereinafter, this activity also called random nicking activity), endonuclease (endonuclease) V it can be mentioned.

Endonuclease V, in vivo, is believed to be the enzyme involved in the DNA repair.

Usually, the endonuclease V is the DN A on Dokishiadenin (dA), caused by deamination, recognizes Dokishiinoshin (d I), in its one base behind in its single-stranded DN A, cut (Nick: nick ) placed, 3 to cause '0H terminus and 5' P terminus is, Kow, the YW groups, studies using E. coli, have been strained clarified (Yao, M. et al., J . Biol. Chem., 1994, 269, 16260- 16268). In addition, the study of after, the endonuclease V, Dokishiuriji

Four

Replacement and paper (Rule 26) down (d U), mismatch site of a double-stranded DNA, further, can have flaps (flap) Nitsu, activity nick been located that observed recognizes this . Heat bacteria Ya thermophiles (hereinafter, also referred to as heat bacteria, etc.) in, there is endonuclease V 'that has been ascertained (for example, about the Therraotoga maritima:. Huang, J. et al, Biochemistry, 2001, 0, 8738-8748). E endonuclease V derived from this Thus to thermophilic bacteria, etc., similar to the E endonuclease V from E. coli, in the presence of magnesium ions, dl, d U, in mismatch site, etc., observed the activity put Knitting click are, further comprising a random nicking activity described above, and, for the single-stranded DN a, DN a nuclease activity, hardly recognized this and force S Akirararyoku strained been in Rereru (Huang, J . et al., Biochemistry, 2001, 40, 738- 8748)

Can be used in the present DNA amplification method, derived endonuclease V is not particularly limited, Escherichia coli, Bacillus subtilis, yeast or the like, can be used those derived from various organisms, endonuclease V from heat bacteria like used, at elevated temperatures (5 0~6 5 ° about C), carrying out the present DN a amplification method, long half-life of the enzymatic activity of the endonuclease V, is suitable.

Resistant bacteria such as the source of E endonuclease V from such heat bacteria etc. is not particularly limited, for example, Archaeoglobus fulgidus (Liu, J. et al.,

Mutation Res., Vol.461, ppl69-177, 2000), Therraoplasraa acidphi 丄觀

(GenBank accession no. AL445064) ^ Pyrococcus abyssi (GenBank accession no. AJ248287), Sulfolobus solfataricus (GenBank accession no. C 002754), Aeropyrus pernix (GenBank accession no. AP000059), or the like can mosquitoes be mentioned. From various organisms, the method of separation 'purified endonuclease V is not particularly limited, it can be performed according to existing isolation and purification methods.

For example, already when the nucleotide sequence of the gene DNA endonuclease V has been identified, using the P CR primers to amplify the DNA, the DNA of the target raw material, amplification of DNA such as a PCR method it demonstrates, after incorporating the DNA amplification production was into a suitable expression vector, introduced into host cells (E. coli strain, etc.), the transformed strain of this, and this to produce to express endonuclease V gene Accordingly, it is possible to perform separation of the endonuclease V. Then, to this crude enzyme, usual known methods, i.e., for example, treatment with a protein precipitant, ultrafiltration, gel filtration, high performance liquid chromatography, centrifugation, electrophoresis, Afi two using a specific antibody tea chromatography dialysis method, by performing purification by conventional methods, can be obtained Endo nuclease V purified to a degree suitable for use in the present DN a amplification.

Also, organisms to be a source of endonuclease V, when the base sequence of a gene DN A endonuclease V has not been identified, for example, the nucleotide sequence of the gene DN A of Endonuku les Aze V is identified It is suitably compares the base Rooster himself column gene DNA of E endonuclease V from multiple closely species, characteristic nucleotide sequence of endonuclease V gene DN a (preferably, to the species It is specific, and, by selecting a base sequence) stored regardless species differences, to prepare a DNA amplification primers it based. Then, using the DNA amplification primers, the subject organism DNA was used as 铸型, using DN A amplification product obtained by DNA amplification method such as P CR method as a probe, the target organisms was prepared by a conventional method DNA from the library one, the endonuclease V of interest can and cloning child. Further, the base sequence of the gene DN A of endonucleases Ichize V of Kuroyungu the subject organism can be determined using conventional methods.

The nucleotide sequence of the determined gene DN A based on, according to the manufacturing method of the endonuclease V using known gene DN A above, still, the base sequence of the gene DN A of endonucleases Ichize V has not been identified of no organisms, it is possible to obtain the endonuclease V.

(2) In the DNA amplification method, as an enzyme (A), all regardless of the E endonuclease such as E endonuclease V with random two Kkingu activity described above, the nick at a specific position of the double-stranded DNA activity that can be put (hereinafter, the activity of this, also referred to as a specific nicking activity) can be used endonuclease having.

Particular can be used in the present DNA amplification method - E down Donuku nuclease having Kkingu activity is not particularly limited.

That is, for example, an endonuclease motor Eve site at which recognizes the base sequence DN A cut away, of the lis type restriction enzyme, those having a specific nicking activity, can be used in the present invention. Among endonucleases with this kind of specific nicking activity, endonuclease obtainable from Bacillus Stearotheartherraophilus the (N. N.BstNBI), it may be mentioned as a suitable endonuclease.

The N. N.BstNBI recognizes the nucleotide sequence GAG ​​TC of double-stranded DN A, an endonuclease having activity nick the four bases under flow (Morgan, RD, et al., Biol. Chera., Vol. 381, pp.1123-1125, 2000).

Moreover, by recognizing the same base sequence as N.BstNBI, an E endonuclease having activity nick, BstSEI (Abdurashitov, MA et al., Ol. Biol., Vol 30, ppl261- 1267, 19% ) it is also one of preferred ones as a endonuclease that have a specific nicking activity used in the present DNA amplification method.

Endonuclease having a specific nicking activity, isolated from the bacteria and the like, the purification method is not particularly limited, and an indicator nicking activity against double-stranded DNA, usually, used methods (above random nicking activity it can be performed Endonuku separation of nuclease, pursuant to purification) with.

In this DN A amplification method, because it exhibits enzyme (A) 1S nicking activity is a requirement, the DNA amplification method, it is Ru necessary der carried out in conditions that satisfy this. As this condition may include, for example, when using an enzyme (A), the presence of magnesium ions.

Ion concentration of this magnesium ions are usually preferred, also a L~1 0 mM, material underlying the magnesium ions are not particularly limited, for example, can be used Ma chloride Guneshiumu, magnesium sulfate, etc. .

2. For enzyme (B)

As an enzyme (B) is typically, 5 '→ 3' Etasonuku nuclease activity include are not DN A polymerase I which permitted.

Such DNA polymerase I, and DNA polymerase activity based on 铸型 DN A, and has a 3 '→ 5' E shit nuclease activity.

Furthermore, the enzyme (B) used in the present DN A amplification method, DNA strand displacement activity

It is preferable that (strand displacement activity) are provided. And the DNA strand displacement activity, on the progress of DNA synthesis by DNA polymerase, the DNA chains attached to the mirror-type DNA strands are present, the existing DNA strand is the activity of replacing the newly synthesized DNA strand (Fujimura, RK et al., J. Biol. Chem., 1976, 251, 2168-2174).

Furthermore, the enzyme (B), it is preferable to 鎳型 DNA exchange activity (template switching activity) are provided.

And the 铸型 DNA exchange activity, the same array or as 鎳型 DNA, or the DN A chain of similar sequence is present, the DNA strand, as 鍚型 DNA instead 鎳型 far, DNA it is active for synthesizing (Odelberg, SJ et al., Nucleic Acids Res., Vol. 25, pp2049-2057, 1995, Guieysee, aL. et al.,

Biochemistry, Vol.34, pp.9193-9199, 1995).

In an enzyme (B), in addition to the DNA polymerase activity, by be provided as described above DNA Kusari置 換活 resistance and 铸型 DNA exchange activity, Te present DNA amplification method odor, a molecular weight of the amplification and the amplified product of DN A it is possible to increase, in performing the amplification of all genes, is a very important factor.

DN A polymerase I that retain activity as described above, already known, there is also commercially available, for example, obtained from E. coli DN A polymerase I, the so-called Tarenou enzymes.

As an enzyme (B) used in the present DNA amplification method, more preferred enzymes are, For example, B st DNA polymerase (large fragment) (Milla, MA et al., Biotechniques, Vol.24, pp.392-396, 1998 ), V ent (exo-) DNA Pojime hydrolase (Kong, H. et al., J. Biol. Chem., Vol.268, pp.1965- 1975, 1993), KODDNA polymerase (Takagi, M. et al. , Appl. Environ. Microbiol., Vol.63, pp4504-4510, 1997), B ca BESTDNA polymerase (Spargo, CA, Mol. Cell. Probes, Vol.10, pp247-256, 1996), ^ 2 9 DNA polymerase over Ze (Blanco, L. et al., J. Biol. Chem., Vol.264, pp.8935-8940, 1989), and the like can be given.

Further, the production method of the enzyme (B) is as possible out be conducted according to conventional known production methods. That is, in the manufacturing method according to the manufacturing method of the enzyme (A), subjected to extraction. Purification from the enzyme derived from the organism of DNA polymerase I. Thereto, for example, performs the appropriate port inlet Teaze treatment, 5 '→ 3' by causing loss of E shit nuclease activity, it is possible to produce the enzyme (B) desired. Furthermore, 5 '→ 3' Ekusonukure Aze activity excluding advance by which gene region responsible for, DN using A polymerase I gene, a host holding an expression vector incorporating the gene DN A, direct enzyme (B) to generate, by performing the extraction and purification of this, it is possible to produce the enzyme (B).

3. aspect of the present DN A amplification method

This DN A amplification method, a double-stranded DN A to be amplified, and said enzyme (A) and (B), by acting against double-stranded DNA of interest, (1) any foregoing hand and two the town DN a with DN a chain and a new DN a chains of either, with respect to the double-stranded DNA formed by a single-stranded DNA each other replaced (2), the enzyme (a ) and reaction with (B), by performing the 違続 manner, amplifies the DN a of interest is a DNA amplification method.

That is, the first view (showing the outline of the DNA amplification method) (1), relative to the double-stranded DNA 1 0 to be amplified, firstly, an enzyme (A) acts, at any position, - click (1 1 1, 1 1 2, 1 1 3) add [FIG. 1 (2)]. As described above, in the process of nick this enters it is required magnesium ions.

Incidentally, the double-stranded DNA 1 0, if double-stranded DNA, is not particularly subject limitation, be a linear DNA, but may be cyclic DNA, prokaryotic or eukaryotic gene such as DN a, double-stranded DNA having a large molecular weight is preferred.

Then these slowly, enzyme (B) (1 2 1, 1 2 2, 1 2 3) from 3'0H the recognized nick, the synthesis of a DNA strand advances in the 3 'direction [Figure 1 ( 3)], this time, DNA chains present on the progress of the synthesis (1 3 1, 1 3 2 1 3 3), the enzyme

The DNA strand displacement activity (B) (strand displacement activity), the new synthetic strand (1 4 1, 1 4 2, 1 4 3) is replaced with [FIG. 1 (4)], exist on the progress of the synthesis Mashimashi DNA strands (1 3 1, 1 3 2 1 3 3), as the substitution proceeds, extends as a single chain D NA [FIG. 1 (5)], where the presence of nicks, these single-stranded DNA, away from the complementary strand.

As shown in FIG. 1, from both sides of the double-stranded DNA 1 0, while performed substituted DNA strand synthesis reaction of DN A progresses. Then, when the new synthetic strand each other intersect, the enzyme (B), more 鍀型 DN A exchange activity (template switching activity), the 鍚型 DNA, changing the DNA strand that is on the synthesis progresses, 鎳型DNA exchange is caused stiffness [e.g., an enzyme (B) 1 2 1 is a 铸型 from DN a chain 1 33, instead of the new synthesized strand 143 to continue the synthesis of chain 14 1, the enzyme (B) 1 2 3 the 铸型 from D NA chain 1 3 1, instead of the new synthetic strand 14 1, you continue synthesis of chains 14 3: FIG. 1 (4)], the new synthetic strand synthesis continues, a new double-stranded DNA is formed [for example, double-stranded DNA consisting of the new synthetic strand 14 1 and 143: FIG. 1 (5)].

Then, the newly synthesized double-stranded DNA, according to the enzyme (A), random Munikkingu is carried out, and the top and the same stroke, further new duplex DN A is synthesized.

- How, (in FIG. 1, the single-stranded DNA 1 3 1, 1 3 2, 1 3 3 corresponds) single-stranded DNA generated by substitution of DN A chains, in the same liquid phase reaction system, having a complementary base sequence to Aniru with other single-stranded DN a. Thus, 'when the end is protruding, by the enzyme (B), 3' 5 of Aniru the DNA DNA synthesis proceeds from the end, the area also becomes double-stranded DNA. Then, also a double-stranded DNA formed in this way, enzymes (A) acts, it has rows of random nicking by stroke as above, again further new double-stranded DNA synthesis as mentioned above being, random nicking by enzymes (a), by successive iterations of replacement by that DNA enzyme (B) synthesis, desired total DN a amplification is efficiently performed.

A series of strokes described above, but illustrates an embodiment where an endonuclease having the enzymatic (A) force random nicking activity, in another embodiment, endonucleases with specific nicking activity, for example, N be used .BstNBI, it can be amplified DN a of interest. In this case, since the position of the DN A that nick entering a specific nucleotide sequence, it is necessary that the base sequence is present a plurality of locations. Thus, in particular, used Endonukure Aze having this particular nicking activity as the enzyme (A), in the aspect of the present DNA amplification method, DNA to be amplified is genomic DNA such prokaryotic or eukaryotic, large molecular weight has, moreover, the double-stranded DN a that there are many endonucleases recognize base sequences used are preferred. Amplification reaction of this DNA is a liquid phase reaction, the reaction solution, and the double-stranded DNA to be amplified, an enzyme (A) enzyme (B) is essential. Wherein the amount ratio in the reaction solution of the enzyme (A) and enzyme (B) is not particularly limited, and the progress of the random nicking by enzymes (A), the initiation of DNA synthesis by the enzyme (B), and, progress, it is preferable to set to be performed with good balance.

The reaction temperature is set according to the optimum temperature of the enzyme (A) and (B) to be used. Ie, an enzyme (A) and (B) force such as E. coli, in the case of biological origin to be viable education in mammalian body temperature (3 7 ° approximately C), the reaction temperature, and 3 7 ° C approximately it is preferred that, as such as heat bacteria, in the case of biological growing at a high temperature (50 to 1 00 ° C position) has a high temperature comparable to the growth environment, such as heat-resistant bacterium of interest it is, generally preferable to. The optimum temperature of the enzyme (A) and (B), it is preferable to choose this in combination with those of the same position of the temperature. For example, the case enzyme (A) is derived from a heat-resistant bacteria such as an enzyme (B) also is suitably a derived heat bacterium.

Further, the reaction p H is also set in accordance with the optimum p H enzyme (A) and (B) to be used. That is, optimum p H enzyme (A) and (B) also, it is preferable to match.

In performing the DNA amplification reactions, as an element to be added to the reaction solution to another, for example, a sufficient amount of d NTP s (deoxynucleotide triphosphates) in DNA synthesis, salts such as N a C 1, enzymes (A) is it include the amount of magnesium I O emissions feed material capable of performing nicking, these, suitable buffers in performing an amplification reaction, for example, by coexisting in Tris-HCl buffer, the DNA amplification reaction It can be performed.

Thus, after the completion of the DN A amplification reaction, the DN A amplification product with purification method according to purification method known PCR amplification reaction products, by performing purification, gene amplification products in accordance with the present DN A amplification method and it can be used as a sample, such as gene analysis.

As one of the points to be noted in performing the DNA amplification method, side reactions like et be.

That, DNA synthesis reaction by DNA polymerase I and the like are in particular conditions, without the addition of DNA and primers as the 鎳型 to the reaction solution, possibility of DNA synthesis is performed have been reported (Ogata, N . and Morino, H., Nucleic Acids Res., Vol.28, pp.3999-4004, 2000, Ogata, N. and Miura, T., Biochemistry, Vol.39, pp.13993-14001, 2000). The good UNA accidental DNA synthesis reactions, in the present DNA amplification method, a side reaction, it is preferable to suppress this, as far as possible.

This side reaction, the reagents in a reaction solution composition, for example, are mixed in the DN A polymerase I, it is believed that stiff force causing the very small amount of short DNA fragments or oligonucleotides.

Therefore, when performing the DNA amplification method, enzyme (A) nor an enzyme (B), it is preferable to use a purified product contamination DN A such as contaminants are eliminated.

4. DN A kit for amplifying

The present invention, Ru invention der also provide kits for performing the DNA amplification method described above.

That is, the present invention, the above (A) and (B), including as a component, for carrying out the present DN A amplification method, DNA amplification kit (hereinafter, this DNA refers kit for amplifying both) to provide it is an invention.

This DNA amplification kit, as essential elements, enzymes (A) and enzyme (B) the content Suruga, other, dNTPs, components for preparing the buffer solution as the base of the reaction solution, feed materials of Ma Guneshiumuion etc., if necessary, it can be included as a component. More specifically, in the examples below, what is used in order to perform the DN A amplification method can be exemplified as a component of the DNA amplification kit.

By using this DN A 增幅 kit, it is possible to perform the DN A amplification method described above efficiently. Example

The following examples further illustrate the present invention. However, more in this embodiment, but the present invention is not limited.

1. Cloning and partial purification of endonuclease V

(1) Cloning

SEQ ID NO: 1, below, is represented by 2, the synthesis of cloning primers for Thermotoga Maritiraa endonuclease V gene full length, was performed according to a conventional method. TMAEND 5 -O: GGG GACA AG TTT GT ACAA AAA AG C AG G CTT CGAAGG AG A TAG AAC CAT GGATTAC AG GCAGCTTCAC AG ATG

(SEQ ID NO: 1)

and

TMAEND- R: GGG GACCAC TTT GTACAA GAA AAG GCC TTT TTT GAG CCG TTG

(SEQ ID NO: 2)

And each of 1 μΜ, as 鑄型 DNA, Thermotoga maritime

Using gene DNA O. Of (ATCC43589D), T br in E XT DNA polymerase hydrolase for PCR kit (first chemical), thermal cycle (9 4 ° C 1 minute, 6 2 ° C 1 minute, 7 2 ° C 1 min) 4 0 cycle repeated. As a result, to obtain a PCR product of a single-band of about 7 0 0 base pairs. This was to confirm the nucleotide sequence of P CR product, was identical to the nucleotide sequence of the published les, Ru Therraotoga maritima E endonuclease V gene (GenBank accession no. A001823).

Then, in this way was purified in a conventional manner to P CR product obtained, Gateway expression The Cloning for downy compactors, p D_〇_NR 2 0 in the input guide according Kit Tomah two Yuanore (Life Technologies one company).

Then, E. coli, Thermotoga maritima Endonuku To nuclease V gene is expressed and the gene expression vector p DEST 1 4, in the kit manual therefore incorporate endonuclease V gene, and used to, E. coli for expression BL the transformation of the 2 1 S 1 share went.

The resulting clones, a composition of L medium except N a C l, the p H 7. 5 with medium conditioned by N a OH, then cultured at 3 7 ° C, to mid-log growth phase, 0. 3M N a C 1 to 5M N a C l to be added, further subjected to culturing for 3 hours, it was carried out induction of the expression of endonuclease V. The bacterial cells were expression induction, and washed with phosphate-buffered saline, and stored at minus 7 0 C.

(2) partially purified

Stroke of partially purified following the E endonuclease V is stroke other than treatment, which describes a process temperature was 0 to 4 ° C. Washed saved cells in lysis buffer (5 0 mM Tris-HCl p H 7. 5, 1 0% sucrose, 5 0 mM N a C 1, 5 raM EDTA, 5 mM 2 ME (2- Mercaptoethanol)) after the bacteria wet weight of about 3 g, it was suspended in lysis buffer for 2 0 ml. It was added Protease Inhibitor cocktail (Roche) in order to suppress the action of proteases thereto. Lysozyme and the reaction was conducted for 0.5 to 1.0 hours was added to a final concentration of 1 MgZml thereto. Next, to a final concentration of 0.1%, was added a 5% Sodium deoxycholate, treated 1 0-3 0 min and lysed. The lysate was centrifuged at 2 OOOOX g, to obtain a supernatant. In the upper rectification, a lysis buffer and force B e 2 OML, further, the 0.5 of 6 ml 1 0% polyrain P (Polyethylenimine) (Sigma) was added to precipitate nucleic acids and acidic proteins were removed by centrifugation . This centrifugal on fine, 5. 4 g of was dissolved by adding sulfuric Anmoniumu, collected by centrifugal sulfate Anmoniumu precipitate proteins, the precipitated proteins, 4 ml of storage buffer (5 0 mM Tris-HCl p H 7. 5 , 5 0 mM N a C 1, I mM EDTA, 1 mM DTT (Dithiothreitol), 5 0% glycerol and (W / V)) was added to dissolve the protein. Then, put this protein lysate in a dialysis tube, Slight 5 0 0 ml of storage buffer Te, was performed twice dialyzed for one 8 hour. Against protein solution was dialyzed, 7 0 ° C 2 0 minutes without line heat treatment, the proteins other than the endonuclease V heat denatured by centrifugal of 1 5 0 0 0 X g, remove heat denatured protein and, the fine on obtained was partially purified products E endonuclease V of Thermotoga maritima.

It should be noted, Thermotoga maritima endonuclease V of purification Kotsure, ί or Te, Huang, H., et al. (Huang, J. et al., Biochemistry, Vol. 40, pp.8738-8748, 2001) have reported force S .

Through process described above, it shows a diagram of a partially purified E endonuclease of V SDS- P AG E (Figure 2). Molecular weight than the above Lane M is a molecular weight marker, respectively, is 9 7 4 0 0, 6 6 0 0 0 4 5 0 0 0 3 1 0 0 0, 2 1 0 0 0 1 4 5 0 0 . Lanes 1 and 2 are each lane corresponding to Therraotoga maritima E endonuclease V which was partially purified from different clonal strain, both the molecular weight was 2 9 0 0 0.

Thus, partially purified products of endonuclease V Themotoga maritima was confirmed that obtained al a. 2. confirmation of nicking activity of the endonuclease V

Covalently linked by ring closure to have double-stranded circular plasmid of DNA, the Nick enters open circular DNA, and the further Both strands same position of closed circular plasmid DNA, when Nick enters a linear DNA Become. These, closed circular, open circular, and linear DNA regardless same molecular weight, respectively, electrophoresis position location in Agarosu electrophoresis are known to be different.

By utilizing this property, obtained by process described above were examined part fraction purified product of DNA nicking activity of the endonuclease V.

That is, the plasmid DNA, and about l Atg the plasmid DNA approximately 4 Kb from p T 7 blue, obtained in the above 1, of the endonuclease V of Theraotoga maritiraa, about l O Ong, nicking activity measurement reaction solution with (20mM Tris- HC1 (p H 7. 5), 20 mM N a C 1, 5mM MgC l 2, ImM DTT) 20 μ 1 in and reacted.

The Figure 3 is the resultant structure by the reaction, 1. loaded onto 2% Agarosugeru, by performing electrophoresis, it is obtained electrophoresis image.

Lane Μ is a lane that corresponds to the molecular weight marker (which was cleavage of the DNA with a restriction enzyme Eta ind III), lane 1 is a lane that corresponds to the controls which do not put endonuclease V, open circular from above plasmid de DNA, linear flop Rasumi de DNA, and bands of closed circular plasmid DN a was present. Lane 2, the reaction temperature of 0 ° C, a lane indicating the result of the reaction for 60 minutes, the concentration of each of the Pando was the same as control. Lane 3, the reaction temperature as 6 5 ° C, a lane shows the results obtained by reacting for 60 minutes.

In FIG. 3, the band indicating the presence of a closed circular plasmid DN A found in control lanes 1 (lowermost band) is lost in lane 3, the in lanes 3 open circular plasmid DN A it has been found that the top of the band shown is darker. The band in the middle indicating the presence of a linear plasmid DN A lane 1 showed the same for both such density lanes 3.

This result, endonuclease V obtained above 1, in 6 5 ° C, revealed that there is activity nick randomly DN A.

3. Amplification of the amplifier (1) Thermus thermophilus gene DNA of DNA

The Thermus thermophilus gene DNA is heat bacteria, separated by a conventional method, which, 1) said first Thermotoga maritima obtained in endonuclease V of 0. 5 μ 1 (l, 2) B st DNA polymerase the 1 μΐ (NEB Inc., 8 units / β ΐ), 1 0 0 μ Μ d Ν Τ Ρ s (1 mM) and MM buffer (Tris - HC1 (p H 1 8. 0), 20 mM N a C 1 , 1 0 raM M g C 1 2, was mixed with ImM DTT (Dithiothreitol)), the total amount was adjusted and the reaction mixture so that Ι Ο Ο μ Ι. DNA amplification reaction, the thus reacted solution was adjusted, having conducted by reacting 1 4 hours at 60 ° C.

After completion of the reaction, using each reaction 1 0 mu 1, having conducted the electrophoresis at 1% Agarosugeru. The electrophoresis image is shown in Figure 4.

In Figure 4, lanes Μ is a lane that corresponds to molecular weight markers,; LD

Shows the H ind II I cleavage product of New Alpha. Lane 1, lane, lane 2 corresponding to controls with no added endonuclease is a lane that corresponds to the control with no added target DNA. In addition, lane 3, the DNA 0.

3 ng, Lane 4, DNA and 3. 0 ng, Lane 5 is DNA a 3 0. 0 ng, Lane 6 is a lane that corresponds to the system by adding respectively 300 ng DNA.

Lane 1 without added endonucleases, and, in lane 2 no added DNA, amplification of DNA was observed. In contrast, lanes 3, 4, the 5 and 6 were added DNA, in all lanes, DNA has been amplified, the amplified DNA is approximately, and a molecular weight of 3 to 0. 5 Kb.

Then, using the Qiagen P CR product purification kit (Qiagen), the amplification product were run in each lane of FIG. 4 and purified, by measuring the OD 2 6 onm, was determined the amount of DNA. The results are shown in Table 1. Table 1 Lane Number 铸型 DNA amount (ng) amplified after DNA amount g) 增幅 magnification (times)

1 30 ND ND

2 0 ND ND

3 3 4. 3 1430

4 30 5.2 170

5 100 8.0 80

6 200 9.1 45

ND: not determined

Amplified DN A quantity initially鎳型DN A weight despite different plus and became almost constant 4~ 9 μ ε / 1 0 0 / ζ 1 reaction.

The causes of certain of the D New Alpha amplification amount, for example, d NT P s is consumed by D NA synthesis 4 hours, DNA synthesis was stopped, but the rate-limiting element etc. are considered normal in the bioreactor is also a recognized is phenomenon, time of DNA amplification reactions in this example, about 4 hours is considered to be efficient.

Further, the purified amplification products, whether or not the amplification products of the gene DNA of Thermus thermophilus is a target DN A becomes a problem. To verify this, focusing on a particular gene, its amplification amount limiting dilution PCR method (Sykes, PJ et. Al., Biothechniques, Vol. 13, pp.444-449, 1992) was performed.

That is, first, the Thermus thermophilus DNA polymerases I gene DNA bases Hai歹 IJ of (GeneBank accesion no.D28878), until 1 0 8 8-1 1 7 8 th, and 1 2 3 1-1 2 6 0 th the two primers having the nucleotide sequence was designed to, these primers, by conventional methods, were synthesized. On the other hand, a known concentration

And dilution steps of 1 0 times the Thermus thermophilus gene DNA, and a 1 0-fold rare dilution stage of amplification products was performed.

To produce a series of PCR reaction solution using two types of primers DN A solution and the performing this dilution, perform PCR method under the same conditions, carried out Agarosugeru electrophoresis on the amplified products by these obtained by comparing the concentration of each Pando after electrophoresis to determine the extent of amplification of the D NA polymerase I gene in product amplified in this D NA amplification method. As a result, the fourth lane 3 of figure roughly 1 0 0 0 fold, lane 4 is approximately 1 0 0-fold, and, lanes 5 and 6 were observed to amplify approximately several tens of times.

Therefore, by using the present DNA amplification method, indeed to be able to amplify the DNA of interest was confirmed.

(2) Amplification of H e L a cellular gene DNA

The study of temporal behavior of the amplification of the DNA by the DNA amplification method was performed using the genomic DNA of H e L a cell is a eukaryotic D NA.

H e L gene DNA 3 0 ng of a cell, placed in the amplification reaction solution (1), 6 0. The amplification reaction was carried out at Flip. 3 every 0 minutes from the start of the reaction, over 2 7 0 minutes, the amount of amplified product according to the D NA amplification method, were purified D NA from the reaction solution by Qiagen PCR product purification kit described above, the OD 2 6 O nm It was calculated by measuring the.

The results, shown in Figure 5. In FIG. 5 the longitudinal axis of a quantity obtained D NA (M g / 1 0 0 μ ΐ), showed the horizontal axis represents time (minutes). The FIG. 5, in the present DN Alpha amplification method, DNA amplification product was found to be obtained by increasing over time. Further, even in eukaryotic gene from DNA, also it revealed that it is possible similarly amplified with prokaryotic gene DNA.

(3) consideration of the case of using a V ent (exo-) DNA polymerase

The DNA amplification of the (1) (2), as a DNA polymerase, was used B st DNA polymerase, even when using another D NA polymerase, it is possible DNA amplification by the present DNA amplification method forces, V ent (exo-) was studied D NA polymerase hydrolase.

That, Thermus maritiraa of E endonuclease V to 1 1 (0. 1 μ g / μ 1), V ent (exo ") DNA polymerase Ichize the 2 mu 1 (NEB, Inc., 2 units / μ 1), 1 0 0 M d NTP s a (1 mM), MM buffer; the (Tris-HCl (p H 8 0), 2 0 mM N a C 1, 1 0 raM M g C 1 2. 1 mM DTT (Dithiothreitol)) mixing, the D NA was added to be cane amplification thereto, the total amount was adjusted to the reaction liquid to be a 1 0 0 μ 1. Next, by amplification reaction of 3 hours 6 5 ° C, the amplified products drawings showing the results of Agarosugeru electrophoresis is a sixth view.

Lane M of Figure 6 are lanes of DNA in H ind III cleavage products with molecular weight markers. Lanes 1 and 2 are the Thermus thermophilus gene DNA a 1 5 ng added was based lanes 增幅 reaction. Lanes 3 and 4 are H e L a cellular gene DNA of 30 ng added was based lanes 增幅 reaction.

As shown in Figure 6, the DN A polymerase, even instead of the V ent (exo ") DNA polymerase, the amplification of DN A that can be performed is confirmed by the DN A amplification.

(4) Examination of the case of using the endonuclease N. B st NBI

Instead of E endonuclease V with random nicking activity, an endonuclease having a specific nicking activity, N. B using st NBI, examine whether the present DNA amplification method are possible amplification of DNA did.

That, 2 OmM Tris-HCl (p H 7. 5), using a reaction solution containing 50 mM KC 1, 1 0 raM M g C 1 2, lmM DTT and l OO ^ M dNTP s, to, 1 μΐ of N. B st NB I (NEB Inc., 1 0unitsZ l) and 1 mu 1 of B st DNA polymerase (NEB Inc., 8 units / μ 1) was added and the DNA further targeted for amplification was added and the reaction the total volume was adjusted to 1 ◦ 0 μ 1.

The amplification reaction was carried out for 3 hours at 6 0 ° C. The resulting amplification product is shown in Figure 7 the results of Agarosugeru electrophoresis.

'Oite in FIG. 7, lane M is a system of lanes of molecular weight markers LDNA of H ind III cleavage products. Lane 1 is a lane of controls with no added DNA to be 增幅 檩的. Lane 2 is a lane of con Toronore with no added ene Donuku nuclease N. B st NB I. Lane 3, thermus

The thermophilus gene DNA is 1 5 ng added was based lane. Then, lane 4, Ru lanes der the same thermus thermophilus gene DNA the system plus 1 50 ng.

As shown in FIG. 7, endonucleases with nicking activity, Or, the absence of added DNA to be amplified is amplified product is not observed, as in lanes 3, 4, DNA and if endonuclease nick is that exist both nicking activity, it is a specific nicking activity, amplification product was found to be be obtained al. Industrial Applicability

The present onset ^, conveniently and efficiently, the full gene amplification means for amplifying the genomic DNA is provided.

Claims

The scope of the claims
1. Two types of enzyme (A) (B):
(A) a double-stranded DNA, the enzyme has an activity capable of nicking; against double-stranded D NA containing the (B) Nick, not nicked, Izu Re or the other D NA chain as 铸型, from the position of the nick, by replacing the side of the DNA strand nicked, to form a new DNA strand complementary base sequence for this 錄型 DN a chain, this 鎳型has a double-stranded DNA to form formed the activity of the DNA strand and the new DNA strands, and are present as a single town DNA replaced by the new D NA strands of said, the nicked side DN degradation of the a chain of the enzyme does not perform; using the amplification step described below, by performing repeatedly in the liquid phase, to amplify the D NA, D NA amplification method.
Amplification step: by acting against subject to double-stranded D NA amplification, the double-stranded DNA with (1) either DNA strand and a new DNA strand of the double-stranded DNA, (2 ), double-stranded D NA formed by single-stranded D NA each other replaced newly allowed made form a these duplexes D NA, as a new amplified DNA, the enzymes (a) and the D NA amplification reaction with (B), the step of performing in succession.
2. an enzyme (A) force endonuclease V, and in the presence of magnesium ions, the reaction is carried out by this enzyme, DNA amplification method of claim 1.
3. Magnesium ion concentration, l to a 1 0RaM, claim 1 or 2 DNA amplification methods described.
4. Enzyme (B), DNA strand displacement activity, and has a 鎳型 DNA strand exchange activity, and, 5 '→ 3' et Taso nuclease activity is not observed, a D NA polymerase peptidase I, claim DNA amplification method according to any one of 1 to 3.
5. Enzyme (A) and Z or (B) is an enzyme derived from thermophilic bacteria or heat bacteria, DN A amplification method in any one of claims 1 to 4.
6. Enzyme {A) or, Thermotoga raaritiraa, Archaeoglobus fulgidus,
Therraoplasma acidphilum, Pyrococcus abyssi, bulf olobus solf ataricus or an endonuclease V from Aeropyrus pernix, DN A amplification method according to any one of claims 1-4.
7. Enzyme (A) ifi, N. BstNBI or a BstSEI, DNA amplification method according to any one of claims 1 to 4.
8. Enzyme (B) force S, B st DNA polymerase (large fragment), V ent (exo-) DNA polymerase, KODDNA polymerase, ψ 2 9 ϋΝ Α polymerase or a B ca BES TDNA polymerase claim: DNA amplification method according to any one of the! ~ 7.
9. with the following enzyme (A) (B):
(A) a double-stranded DNA, an enzyme having the activity capable of nicking
(B) with respect to double-stranded DNA nicked, not nicked, as 铸型 Izu Re or the other DNA strand, the position of the nick, by replacing the side of the DNA strand nicked , to form a new DNA strand complementary base sequence for this 鎳型 DNA strand, the 铸型 DN a chain and a new DN a duplex DN a activity that form the strand Yes and, and, present as a single strand DN a is replaced with a new DNA strand of the degradation of the side of the DNA strand nicked does not perform enzyme
And it includes as a component, for doing one of the DNA amplification method of claim 1-8, kit for DNA amplification.
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Publication number Priority date Publication date Assignee Title
US7632667B2 (en) 2005-10-24 2009-12-15 Nishikawa Rubber Co., Ltd. Mutan endonuclease with substrate-specific cleavage activity
JP2010029089A (en) * 2008-07-28 2010-02-12 Nishikawa Rubber Co Ltd Thermostable endonuclease v

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Publication number Priority date Publication date Assignee Title
EP0497272A1 (en) * 1991-01-31 1992-08-05 Becton Dickinson and Company Strand displacement amplification

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EP0497272A1 (en) * 1991-01-31 1992-08-05 Becton Dickinson and Company Strand displacement amplification

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7632667B2 (en) 2005-10-24 2009-12-15 Nishikawa Rubber Co., Ltd. Mutan endonuclease with substrate-specific cleavage activity
JP2010029089A (en) * 2008-07-28 2010-02-12 Nishikawa Rubber Co Ltd Thermostable endonuclease v

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