CN1810990A

CN1810990A - Method for amplifying nucleic acid sequence

Info

Publication number: CN1810990A
Application number: CN 200510091549
Authority: CN
Inventors: 向井博之; 山本纯子; 武田理; 三宅一惠; 上森隆司; 佐藤好美; 森山麻里子; 椹木治久; 萩屋道雄; 浅田起代蔵; 加藤郁之进
Original assignee: Takara Bio Inc
Current assignee: Takara Bio Inc
Priority date: 1999-03-19
Filing date: 2000-03-14
Publication date: 2006-08-02
Anticipated expiration: 2020-03-14
Also published as: CN100432237C

Abstract

The present invention provides a convenient and effective method for amplifying a nucleic acid sequence characterized by effecting a DNA synthesis reaction in the presence of chimeric oligonucleotide primers; a method for supplying a large amount of DNA amplification fragments; an effective method for amplifying a nucleic acid sequence by combining the above method with another nucleic acid sequence amplification method; a method for detecting a nucleic acid sequence for detecting or quantitating a microorganism such as a virus, a bacterium, a fungus or a yeast; and a method for detecting a DNA amplification fragment obtained by the above method in situ.

Description

The method that is used for amplifying nucleic acid sequence

The application is to be on March 14th, 2000 applying date, and application number is 00807534.4, and denomination of invention is divided an application for the application for a patent for invention of " method that is used for amplifying nucleic acid sequence ".

Technical field

The present invention relates to the method for synthetic DNA, it can be used for the genetically engineered field.It relates to amplification as the method for the nucleotide sequence of template and the method that detects the nucleic acid of described method amplification.

Background technology

The synthetic various purposes that are used for the genetically engineered area research of DNA.Except such as the short chain DNA of oligonucleotide synthetic, most of DNA are synthetic to be finished by the Enzymology method that wherein uses archaeal dna polymerase.An Enzymology method example is polymerase chain reaction (PCR) method, and it is described in detail in U.S. Patent number 4,683,195,4,683,202 and 4,800,159.Another example is reverse transcription PCR (RT-PCR) method, its associating PCR method and reverse transcriptase reaction, and Trends in Biotechnology for example, 10:146-152 (1992) is described.But the purpose district of feasible DNA amplification of the development of aforesaid method or RNA.

For example adopt the reaction of 3 steps to carry out above-mentioned DNA synthesis method.3 steps are as follows: step be make double-stranded DNA dissociate (sex change) be single stranded DNA, a step is to make primer annealing to described single stranded DNA, a step is the complementary strand of synthetic (prolongations) described primer, so that increase the target DNA district.Perhaps, employing is called " PCR shuttles back and forth " (" PCR hosaizensen " (Recent advances in PCR methodology:Basic methodology andit ' s application), Tanpakushitsu Kakusan Kouso, Bessatsu, (Protein, Nucleic Acid and Enzyme, supplementary issue), 41 (5): reaction 425-428 (1996)) is carried out, 2 steps in wherein said 3 steps, and promptly annealing primer and prolongation step are carried out under uniform temp.

Perhaps, can use as EP 320,308 described ligase chain reaction (LCR) (LCR) methods of announcement on June 14th, 1989 or as PCR Protocols, Academic Press Inc., 1990, the 245-252 pages or leaves are described based on the amplification system of transcribing (TAS) method.Above-mentioned 4 kinds of methods need be under high temperature and low temperature reaction repeated repeatedly so that produce the strand target molecule that is used for next amplification cycles again.Should adopt discontinuous phase or circulation to carry out described reactive system, because this reaction-limited is in temperature as mentioned above.

Therefore, described method need adopt and can strict regulate the expensive thermal cycler of wide region temperature in time.And the described reaction needed time is with temperature regulation to 2 or 3 preset temperatures.Expend time in and be directly proportional with cycle index.

In order to solve described problem, but developed the method for carrying out nucleic acid amplification under the homothermic situation.The example comprises that the described strand displacement amplification of JP-B 7-114718 (SDA) method, oneself continue sequence replicating (3SR) method, Japanese Patent No. 2650159 described amplification based on nucleotide sequence (NASBA) methods, amplification (TMA) method of transcriptive intermediate, Japanese Patent No. 2710159 described Q β replicative enzyme method and U.S. Patent numbers 5,824,517, WO 99/09211, WO 95/25180 and WO 99/49081 described various improvement SDA methods.U.S. Patent number 5,916,777 have introduced the method for constant temperature zymetology synthetic oligonucleotide.In the reaction of the method for these constant temperature nucleic acid amplifications or synthetic oligonucleotide, parallelly in the reaction mixture of constant temperature incubation carry out primer extension and/or on the product that primer annealing behind the primer extension prolongs to strand (or initial target sequence).

In described constant temperature nucleic acid amplification method, the SDA method is the system example of wherein final DNA amplification.The SDA method is to utilize the double-stranded and method of target nucleic acid sequence (with its complementary strand) in the amplification sample of archaeal dna polymerase and restriction endonuclease displacement.Four kinds of primers that this method need be used to increase; Wherein two kinds of primers should be designed to contain described restriction endonuclease recognition site.For a large amount of synthetic DNAs, this method need be with the triphosphate deoxyribose nucleotide of modifying as substrate.The triphosphate deoxyribose nucleotide example of a modification is the (triphosphate deoxyribose nucleotide of α-S) that the α position Sauerstoffatom of wherein phosphate is replaced by sulphur atom (S).If for example in order to carry out genetic experiment, routine is carried out described reaction, then the cost problem of following with the triphosphate deoxyribose nucleotide of use modifying becomes serious problem.In addition, for example when the Nucleotide that the dna fragmentation that is increased is carried out Restriction Enzyme fragment length polymorphism (RFLP) when analyzing, in the dna fragmentation that described method increased, add modifying as (but the deoxyribonucleotide of α-S) may be eliminated the cutting of Restriction Enzyme to the dna fragmentation that increased.

U.S. Patent number 5,824, the 517 improvement SDA methods of being introduced are a kind of DNA cloning method, this method is used the chimeric primers of RNA and DNA composition and is had the structure that is positioned at 3 '-end as the wherein DNA of primary element at least.The improvement SDA method that WO 99/09211 introduces need be used the restriction enzyme that produces 5 '-protruding terminus.WO 95/25180 described improvement SDA method need be used at least 2 pairs of primers.The improvement SDA method that WO 99/49081 introduces need be used the triphosphate deoxyribose nucleotide of at least 2 pairs of primers and at least a modification.On the other hand, U.S. Patent number 5,916, the method of 777 synthetic oligonucleotides of introducing comprises that utilization has the primer synthetic DNA of ribonucleotide at 3 '-end, use described primer to finish reaction, import one with its breach that separates between the primer of primer-extended chain and extended chain with endonuclease, the digestion template also reclaims recycling primer.Need separate described primer from described reactive system, it is annealed on the template again, in described method, to utilize described primer again.

As mentioned above, still there is variety of issue in described conventional constant temperature nucleic acid amplification method.Therefore, need the method for low-cost amplification of nucleotide acid sequence, can obtain the further dna fragmentation of genetic engineering procedure with such method.

Goal of the invention

Main purpose of the present invention provides the simple and effective method of amplification of nucleotide acid sequence, it is characterized by and carry out synthesis reaction of DNA in the presence of Oligonucleolide primers, and the present invention also provides the method for mass production amplification of DNA fragments, with the supply amplification of DNA fragments.

Summary of the invention

Because carried out deep research, so the inventor has made up the good system that is used for gene amplification reaction.Develop a kind of method in target DNA district that increases at 3 '-end or in the presence of the chimeric oligonucleotide primer that has ribonucleotide on 3 '-end side, endonuclease and archaeal dna polymerase, thereby finish the structure of described good system.Therefore, finished the present invention.This method of the present invention is called the method that constant temperature uses chimeric primers amplification of nucleic acid (ICAN), and it is a kind of method of using the amplification of nucleotide acid sequence of chimeric oligonucleotide primer under constant temperature.

First invention of the present invention relates to the method that is used for the amplification of nucleotide acid sequence, it is characterized in that described method comprises:

(a) with basic complementary primer of the nucleotide sequence of at least a and described nucleic acid and archaeal dna polymerase processing template nucleic acid, with synthetic and template complementary primer extension chain, wherein said primer is the chimeric oligonucleotide primer that comprises deoxyribonucleotide and ribonucleotide, described ribonucleotide is positioned on 3 '-end side of 3 ' of primer-end or primer, so that cut with endonuclease;

(b) with the primer extension chain of described endonuclease at the double-strandednucleic acid of site cutting step (a) acquisition that comprises described ribonucleotide; And

(c) with having the active archaeal dna polymerase of strand displacement, from primer 3 '-terminal the extension and template complementary nucleotide sequence partly of described double-strandednucleic acid, wherein the primer extension chain of cutting step (b) acquisition reaches strand displacement.

Second invention of the present invention relates to the method with at least two kinds of primer amplification nucleotide sequences, it is characterized in that described method comprises:

(b) with the primer extension chain of described endonuclease at the double-strandednucleic acid of site cutting step (a) acquisition that comprises described ribonucleotide;

(c) with having the active archaeal dna polymerase of strand displacement, primer 3 '-terminal the extension and template complementary nucleotide sequence partly from described double-strandednucleic acid, wherein the primer extension chain of cutting step (b) acquisition reaches strand displacement, and wherein step (b) is reused the double-strandednucleic acid that contains the primer extension chain that produces again;

(d) with at least a primer of step (a) use and the release displacement chain template of archaeal dna polymerase treatment step (c) acquisition of being different from, with synthetic and displacement chain complementary primer extension chain, wherein be different from the primer that step (a) uses and be chimeric oligonucleotide primer, this primer is with the basic complementation of the nucleotide sequence of displacement chain and contain deoxyribonucleotide and ribonucleotide, described ribonucleotide is positioned on 3 '-end side of 3 ' of primer-end or primer, so that cut with endonuclease;

(e) with the primer extension chain of endonuclease at the double-strandednucleic acid of site cutting step (d) acquisition that contains ribonucleotide; And

(f) with having the active archaeal dna polymerase of strand displacement, primer 3 '-terminal the extension and template complementary nucleotide sequence partly from described double-strandednucleic acid, wherein the primer extension chain of cutting step (e) acquisition reaches strand displacement, and wherein step (e) is reused the double-strandednucleic acid that contains the primer extension chain that produces again.

Carry out first inventive method of the present invention and second inventive method under the constant temperature.The template nucleotide sequence can be a dna sequence dna.The step (a) of described first invention and second invention can comprise the step for preparing strand cDNA with ThermoScript II and template ribonucleic acid by reverse transcription reaction before.Described strand cDNA can be used as the template nucleotide sequence.Single stranded DNA and double-stranded DNA all can be used as template DNA in first invention of the present invention and second invention.If double-stranded DNA is as template, then can after the pre-treatment step that to make described double-stranded DNA sex change be single stranded DNA, implement method of the present invention.

In above-mentioned the present invention, carry out the extension of described primer with having the active archaeal dna polymerase of strand displacement.The Bca archaeal dna polymerase that is selected from the shortage 5 '-3 ' exonuclease of the BstDNA polysaccharase of shortage 5 '-3 ' exonuclease of Klenow fragment, bacstearothermophilus (Bacillus stearothermophilus) of e. coli dna polymerase I and hot hard genus bacillus (Bacillus caldotenax) is preferred for the present invention.In addition, endoribonuclease can be preferably used as endonuclease.Spendable endoribonuclease includes but not limited to for example RNA enzyme H.

The 3rd invention of the present invention relates to the method for amplification of nucleotide acid sequence, it is characterized in that described method comprises:

(a) by hybrid template nucleic acid, triphosphate deoxyribose nucleotide, have an endonuclease preparation feedback mixture of the extended chain that the active archaeal dna polymerase of strand displacement, at least a primer and cutting primer produce, wherein said primer is for basic complementary and comprise the chimeric oligonucleotide primer of deoxyribonucleotide and ribonucleotide with the nucleotide sequence of template nucleic acid, described ribonucleotide is positioned on 3 '-end side of 3 ' of primer-end or primer, so that cut with endonuclease; And

(b) the enough time of incubation reaction mixture is to produce reaction product.

The example of spendable template nucleotide sequence comprises the nucleotide sequence of the cDNA that the reverse transcription reaction that is selected from double-stranded DNA that single stranded DNA, sex change are single stranded DNA and RNA obtains in the 3rd invention of the present invention.In described reaction mixture, may comprise the chimeric oligonucleotide primer more than 2 kinds or 2 kinds.First invention and the second invention use have the active archaeal dna polymerase of strand displacement and endonuclease can be preferred for the present invention.

The primer that uses in first invention of the present invention is invented to the 3rd is chimeric oligonucleotide primer.For example can use to have wherein and connect at least 1, the chimeric oligonucleotide of the structure of preferred continuous kernel sugar nucleotide residue more than 2 or 2 on 3 ' of described primer-end or 3 '-end side.

The template of using in first to the 3rd invention of the present invention can be the nucleic acid that increases with nucleic acid amplification method in advance.Although can use the method for any amplification of nucleic acid ad lib, for example TAS method, 3SR method, NASBA method, TMA method, Q β replicative enzyme method, PCR method, LCR method and SDA method can be used as nucleic acid amplification method.Method of the present invention can be united use with these nucleic acid amplification methods.

In nucleic acid amplification reaction, can use random primer or degenerated primer.For example can preferably use ad lib at least 3 '-terminal or on 3 '-end side, have a primer of stochastic sequence or degenerate sequence.

The 4th invention of the present invention relates to and can be used for the chimeric oligonucleotide primer of first invention to the 3rd invention.Being characterized as it and comprising deoxyribonucleotide and ribonucleotide and have wherein that ribonucleotide is positioned at 3 ' of primer-end or the structure on 3 ' of primer-end side of this primer.For example can use a kind of like this chimeric oligonucleotide primer: it comprise at least one, preferred 2 or 2 above continuous kernel sugar nucleotide residues and can be from its 3 ' terminal extended DNA chain.Design such primer, so that act on 3 ' of ribonucleotide residue-end and cut described primer such as the rnase of RNA enzyme H.

The 5th invention of the present invention relates to and is used for having the active archaeal dna polymerase of strand displacement, endonuclease and comprising their test kit of first to the 3rd invention.

The 6th invention of the present invention relates to the method that detects target nucleic acid, it is characterized in that described method comprises the method amplifying target nucleic acid of of the present invention first to the 3rd nucleotide sequence of inventing that is used to increase, and detects described nucleic acid then.Detection method comprises the method for ribonucleotide (RNA) the probe in detecting target nucleic acid of 2 or 2 above fluorochrome labels that wherein are in quencher state distance.

The 7th invention of the present invention relates to the having the active archaeal dna polymerase of strand displacement, endonuclease and comprise their test kit of method of the target nucleic acid that is used to detect the present invention's the 6th invention.

The 8th invention of the present invention relates to the method that preparation comprises the material of fixed nucleic acid, wherein said nucleic acid layout is characterized in that in prospective region described method comprises that the nucleic acid arrangement that the method with of the present invention first to the 3rd amplification of nucleotide acid sequence of inventing is increased is fixed on the prospective region on the upholder.Especially preferably a kind of like this method: increase and the basic free single-chain nucleic acid of its complementary strand in the method, and its arrangement is fixed in the prospective region.

The 9th invention of the present invention relates to the material with fixed nucleic acid, and wherein said nucleic acid layout is in the prospective region that the method with the 8th invention of the present invention prepares.The material that especially preferably has fixed nucleic acid is wherein arranged with the basic free single-chain nucleic acid of its complementary strand and is fixed on prospective region.

The tenth invention of the present invention relates to the method for the target nucleic acid in the test sample, it is characterized in that described method use has the material of fixed nucleic acid, wherein said nucleic acid layout is in the prospective region of the 9th invention of the present invention, with the nucleic acid of the arrangement fixed nucleic acid hybridization in detection and the described material prospective region.

The 11 invention of the present invention relates to the method for mass production nucleic acid, it is characterized in that described method comprises:

(a) with basic complementary primer of the nucleotide sequence of at least a and described nucleic acid and archaeal dna polymerase processing template nucleic acid, with synthetic and template complementary primer extension chain, wherein said primer is the chimeric oligonucleotide primer that comprises deoxyribonucleotide and ribonucleotide, described ribonucleotide is positioned on 3 '-end side of 3 ' of described primer-end or primer, so that cut with endonuclease;

(b) with the primer extension chain of endonuclease in the double-strandednucleic acid of site cutting step (a) acquisition that comprises ribonucleotide; And

(c) with having the active archaeal dna polymerase of strand displacement from 3 ' of double-strandednucleic acid primer part-terminal extension and template complementary nucleotide sequence, wherein the primer extension chain that obtains of cutting step (b) is to reach strand displacement.

The 12 invention of the present invention relates to the method that produces nucleic acid with at least two kinds of primers in a large number, it is characterized in that described method comprises:

(d) with at least a primer of step (a) use and the release displacement chain template of archaeal dna polymerase treatment step (c) acquisition of being different from, with synthetic and displacement chain complementary primer extension chain, wherein be different from the described primer that step (a) uses and be chimeric oligonucleotide primer, this primer is with the basic complementation of the nucleotide sequence of displacement chain and contain deoxyribonucleotide and ribonucleotide, described ribonucleotide is positioned on 3 '-end side of 3 ' of primer-end or primer, so that cut with endonuclease;

The 13 invention of the present invention relates to the method for mass production nucleic acid, it is characterized in that described method comprises:

The 14 invention of the present invention relates to the method for amplification of nucleotide acid sequence, it is characterized in that described method comprises:

(a) comprise the nucleic acid of the sequence of needs amplification by nucleic acid amplification reaction amplification, to prepare template nucleic acid;

(b) template nucleic acid that obtains with basic complementary primer of the nucleotide sequence of at least a and described nucleic acid and archaeal dna polymerase treatment step (a), with synthetic and template complementary primer extension chain, wherein said primer is the chimeric oligonucleotide primer that comprises deoxyribonucleotide and ribonucleotide, described ribonucleotide is positioned on 3 '-end side of 3 ' of primer-end or primer, so that cut with endonuclease;

(c) with the primer extension chain of described endonuclease at the double-strandednucleic acid of site cutting step (b) acquisition that comprises described ribonucleotide; And

(d) with having the active archaeal dna polymerase of strand displacement, from primer 3 '-terminal the extension and template complementary nucleotide sequence partly of described double-strandednucleic acid, wherein the primer extension chain of cutting step (c) acquisition reaches strand displacement.

The 15 invention of the present invention relates to the method with at least two kinds of primer amplification nucleotide sequences, it is characterized in that described method comprises:

(c) with the primer extension chain of described endonuclease at the double-strandednucleic acid of site cutting step (b) acquisition that comprises described ribonucleotide;

(d) with having the active archaeal dna polymerase of strand displacement, from primer 3 '-terminal the extension and template complementary nucleotide sequence partly of described double-strandednucleic acid, wherein the primer extension chain of cutting step (c) acquisition reaches strand displacement; Wherein step (c) is reused the double-strandednucleic acid that contains the primer extension chain that produces again;

(e) with at least a primer of step (b) use and the release displacement chain template of archaeal dna polymerase treatment step (d) acquisition of being different from, with synthetic and displacement chain complementary primer extension chain, wherein be different from the described primer that step (b) uses and be chimeric oligonucleotide primer, this primer is with the basic complementation of the nucleotide sequence of displacement chain and contain deoxyribonucleotide and ribonucleotide, described ribonucleotide is positioned on 3 '-end side of 3 ' of primer-end or primer, so that cut with endonuclease;

(f) with the primer extension chain of endonuclease at the double-strandednucleic acid of site cutting step (e) acquisition that contains ribonucleotide; And

(g) with having the active archaeal dna polymerase of strand displacement, primer 3 '-terminal the extension and template complementary nucleotide sequence partly from described double-strandednucleic acid, wherein the primer extension chain of cutting step (f) acquisition reaches strand displacement, and wherein step (f) is reused the double-strandednucleic acid that contains the primer extension chain that produces again.

The 16 invention of the present invention relates to the method for amplification of nucleotide acid sequence, it is characterized in that described method comprises:

(b) template nucleic acid, the triphosphate deoxyribose nucleotide that obtains by mixing step (a), have the endonuclease preparation feedback mixture of the extended chain that the active archaeal dna polymerase of strand displacement, at least a primer and cutting primer produce, wherein said primer is for basic complementary and comprise the chimeric oligonucleotide primer of deoxyribonucleotide and ribonucleotide with the nucleotide sequence of template nucleic acid, described ribonucleotide is positioned on 3 '-end side of 3 ' of primer-end or primer, so that cut with endonuclease; And

(c) the enough time of incubation reaction mixture is to produce reaction product.

In the 14 to the 16 invention of the present invention, increasing in advance by nucleic acid amplification reaction comprises the nucleic acid of the sequence that needs increase.Amplified production is as the template nucleic acid in first to the 3rd inventive method of the present invention then.Although can use the method for any amplification of nucleic acid ad lib, for example TAS method, 3SR method, NASBA method, TMA method, Q β replicative enzyme method, PCR method, LCR method and SDA method can be used as nucleic acid amplification method in the 14 to the 16 invention.

The 17 invention of the present invention relates to the method for amplification of nucleotide acid sequence, it is characterized in that described method comprises:

(a) with basic complementary primer of the nucleotide sequence of at least a and described nucleic acid and archaeal dna polymerase processing template nucleic acid, with synthetic and template complementary primer extension chain, wherein said primer is the chimeric oligonucleotide primer that comprises deoxyribonucleotide and ribonucleotide, and described ribonucleotide is positioned on 3 '-end side of 3 ' of primer-end or primer;

(b) with the primer extension chain of endonuclease at the double-strandednucleic acid of site cutting step (a) acquisition that comprises described ribonucleotide; And

The 18 invention of the present invention relates to the method with at least two kinds of primer amplification nucleotide sequences, it is characterized in that described method comprises:

(b) with the primer extension chain of endonuclease at the double-strandednucleic acid of site cutting step (a) acquisition that comprises described ribonucleotide;

(d) with at least a primer of step (a) use and the release displacement chain template of archaeal dna polymerase treatment step (c) acquisition of being different from, with synthetic and displacement chain complementary primer extension chain, wherein be different from the primer that step (a) uses and be chimeric oligonucleotide primer, this primer is with the basic complementation of the nucleotide sequence of displacement chain and contain deoxyribonucleotide and ribonucleotide, and described ribonucleotide is positioned on 3 '-end side of 3 ' of primer-end or primer;

The 19 invention of the present invention relates to the method for amplification of nucleotide acid sequence, it is characterized in that described method comprises:

(a) by hybrid template nucleic acid, triphosphate deoxyribose nucleotide, have an endonuclease preparation feedback mixture of the extended chain that the active archaeal dna polymerase of strand displacement, at least a primer and cutting primer produce, wherein said primer is for basic complementary and comprise the chimeric oligonucleotide primer of deoxyribonucleotide and ribonucleotide with the nucleotide sequence of template nucleic acid, and described ribonucleotide is positioned on 3 '-end side of 3 ' of primer-end or primer; And

The 20 invention of the present invention relates to the method for amplification of nucleotide acid sequence, it is characterized in that described method comprises:

(a) with basic complementary primer of the nucleotide sequence of at least a and described nucleic acid and archaeal dna polymerase processing template nucleic acid, with synthetic and template complementary primer extension chain, wherein said primer is the chimeric oligonucleotide primer that comprises deoxyribonucleotide and ribonucleotide, described ribonucleotide is positioned on 3 '-end side of 3 ' of described primer-end or primer, and wherein endonuclease cuts in the site that comprises ribonucleotide;

(b) with the primer extension chain of described endonuclease in the double-strandednucleic acid of site cutting step (a) acquisition that comprises ribonucleotide; And

The 21 invention of the present invention relates to the method with at least two kinds of primer amplification nucleotide sequences, it is characterized in that described method comprises:

(b) with the primer extension chain of described endonuclease in the double-strandednucleic acid of site cutting step (a) acquisition that comprises ribonucleotide;

(c) with having the active archaeal dna polymerase of strand displacement from 3 ' of double-strandednucleic acid primer part-terminal extension and template complementary nucleotide sequence, wherein the primer extension chain of cutting step (b) acquisition is to reach strand displacement, and wherein step (b) is reused the double-strandednucleic acid that contains the primer extension chain that produces again;

(d) with at least a primer of step (a) use and the release displacement chain template of archaeal dna polymerase treatment step (c) acquisition of being different from, with synthetic and displacement chain complementary primer extension chain, wherein be different from the primer that step (a) uses and be chimeric oligonucleotide primer, this primer is with the basic complementation of the nucleotide sequence of displacement chain and contain deoxyribonucleotide and ribonucleotide, described ribonucleotide is positioned on 3 '-end side of 3 ' of primer-end or primer, and wherein endonuclease cuts in the site that comprises ribonucleotide;

(e) with the primer extension chain of described endonuclease at the double-strandednucleic acid of site cutting step (d) acquisition that contains ribonucleotide; And

The 22 invention of the present invention relates to the method for amplification of nucleotide acid sequence, it is characterized in that described method comprises:

(a) by hybrid template nucleic acid, triphosphate deoxyribose nucleotide, have an endonuclease preparation feedback mixture of the extended chain that the active archaeal dna polymerase of strand displacement, at least a primer and cutting primer produce, wherein said primer is for basic complementary and comprise the chimeric oligonucleotide primer of deoxyribonucleotide and ribonucleotide with the nucleotide sequence of template nucleic acid, described ribonucleotide is positioned on 3 '-end side of 3 ' of primer-end or primer, and wherein said endonuclease cuts in the site that comprises described ribonucleotide; And

The 23 invention of the present invention relates to the method for the nucleotide sequence of measuring nucleic acid, it is characterized in that described method comprises the method amplification of nucleotide acid sequence according to arbitrary invention in first to the 3rd invention of the present invention and the 14 to the 22 invention.

The accompanying drawing summary

Fig. 1 is the schema that an example of the inventive method of wherein using strand is described.In the figure, the released dna chain that indicates with solid garden is used as the template DNA in (6).

Fig. 2 demonstration utilizes the agarose gel electrophoresis result of differential responses time through the dna fragmentation of method amplification of the present invention.

Detailed Description Of The Invention

Deoxyribonucleotide used herein (being also referred to as dN) refers to the nucleotides that its sugar moieties is made of the D-2-deoxyribose. Deoxyribonucleotide comprises that for example base portion is the deoxyribonucleotide of adenine, cytimidine, guanine or thymidine.

Ribonucleotide used herein (being also referred to as N) refers to the nucleotides that its sugar moieties is made of D-ribose. Ribonucleotide comprises that base portion is the ribonucleotide of adenine, cytimidine, guanine or uracil. Described ribonucleotide also comprises the ribonucleotide of modification, and for example wherein the modification ribonucleotide that replaced by sulphur atom of the oxygen atom of the phosphate put of alpha-position (is also referred to as (ribonucleotide of α-S) or (α-S) N) or other derivative.

The chimeric oligonucleotide primer that the present invention uses refers to comprise the primer of deoxyribonucleotide and ribonucleotide. Described primer can comprise the deoxyribonucleotide of unmodified and/or modify deoxyribonucleotide. Perhaps it can comprise the unmodified ribonucleotide and/or modify ribonucleotide.

Be used for chimeric oligonucleotide primer of the present invention comprise any described primer 3 '-end or 3 ' of described primer-end side have ribonucleotide chimeric oligonucleotide primer, can be used for extending in the methods of the invention nucleic acid chains, available endonuclease cuts and can be used for realizing strand replacement reaction.

3 '-end side used herein refers to the center to 3 such as the nucleic acid of primer '-terminal part. Equally, 5 '-end side refers to the center to 5 of nucleic acid '-terminal part.

Endonuclease used herein can be act on the double-stranded DNA that obtained by the chimeric oligonucleotide primer extended DNA that is annealed on the template nucleic acid and also in the primer part specificity that comprises ribonucleotide to its any endonuclease that cuts.

Archaeal dna polymerase used herein refers to utilize the again enzyme of synthetic DNA chain of template DNA chain. Described archaeal dna polymerase include but not limited to pol I-type archaeal dna polymerase (for example e. coli dna polymerase I, Klenow fragment and Taq archaeal dna polymerase), α-type archaeal dna polymerase (for example archaeal dna polymerase of fierce hot-bulb bacterium (Stratagene), VENT archaeal dna polymerase (New England Biolabs), KOD archaeal dna polymerase (Toyobo) and DEEP VENT archaeal dna polymerase (New England Biolabs)) and non-α-, non--pol I-type archaeal dna polymerase (for example WO 97/24444 introduce archaeal dna polymerase). Archaeal dna polymerase with strand displacement activity comprises Thermophilic Bacteria such as the archaeal dna polymerase of Bacillus caldotenax (after this being called B.ca) and bacillus stearothermophilus (after this being called B.st) and the variant that these archaeal dna polymerases lack its 5 '-3 ' exonuclease activity of bacillus. And strand displacement type archaeal dna polymerase comprises the archaeal dna polymerase that has the strand displacement activity and do not have 5 '-3 ' exonuclease activity, for example Klenow fragment. In addition, described archaeal dna polymerase can be the mixture of multiple archaeal dna polymerase, for example has the archaeal dna polymerase of strand displacement activity and does not have the hard-core mixture of the archaeal dna polymerase of strand displacement activity.

" strand displacement is active " used herein refers to realize the activity of strand displacement, namely can carry out dna replication dna according to the nucleotide sequence of template, replaces simultaneously described DNA chain, is annealed to the complementary strand of template strand with release. In addition, because strand displacement is referred to herein as " release chain " from the DNA chain that the template nucleotide sequence discharges.

Introduce in more detail below the present invention:

(1) is used for chimeric oligonucleotide primer of the present invention

The primer that uses in the method for the invention is the chimeric oligonucleotide primer that comprises deoxyribonucleotide and ribonucleotide. Such primer comprises the Oligonucleolide primers that comprises the unmodified ribonucleotide and/or modify ribonucleotide.

The chimeric oligonucleotide primer that uses in the method for the invention can be any such primer: have with the basic complementary nucleotide sequence of a part of nucleotide sequence of template nucleic acid, can be used for having the site that endonuclease cuts synthesis reaction of DNA from its 3 '-terminal extended DNA chain and at its 3 '-end or 3 '-end side. For example can use 3 '-terminal or 3 '-end side has the chimeric oligonucleotide primer of ribonucleotide. Usually the described primer of design is so that it and a part that needs amplification section upstream (part that namely is equivalent to the nucleotide sequence 3 ' of the section that needs increase in template nucleic acid) complementation. " basic complementary nucleotide sequence " used herein refers to can be annealed to the nucleotide sequence of template DNA under used reaction condition. Those skilled in the art for example can design this chimeric oligonucleotide primer or Oligonucleolide primers with reference to Labo Manual PCR (Takara Shuzo, 13-16 page or leaf, 1996) according to known method. Can use the commercial software of design primer, for example OLIGO^TMPrimer analysis software (Takara Shuzo).

The chimeric oligonucleotide primer that uses in the method for the invention can comprise one or more and modify ribonucleotide. Ribonucleotide used herein can be the ribonucleotide of unmodified or modify ribonucleotide that they can be positioned on 3 ' of chimeric oligonucleotide primer-end or 3 '-end side and be identified by endonuclease or cut by endonuclease. Described ribonucleotide comprises aforesaid unmodified ribonucleotide and modifies ribonucleotide. Unmodified ribonucleotide or modification ribonucleotide or its combination can be used for chimeric oligonucleotide primer of the present invention, and condition is the function that it does not eliminate primer. The example of modifying ribonucleotide include but not limited to the oxygen atom of wherein being combined with phosphate by sulphur atom replace (hydroxyl of the ribonucleotide of α-S) and ribose 2-position is by the ribonucleotide of methoxy substitution. For example use vulcanization reaction reagent (Glen Research) or 2-OMe-RNA-CE phosphoramidite reagent (Glen Research) through U.S. Patent number 5 by adopting, 003, the 097 method preparation of introducing (α-S) the triphosphoric acid ribonucleotide can produce this chimeric oligonucleotide primer of modifying ribonucleotide that comprises.

Can design the chimeric oligonucleotide primer that can use and comprise the modification ribonucleotide in amplification method of the present invention, it gives the resistance to the endonuclease cutting. This primer is useful, because people can be controlled in the site of cutting with endonuclease in the amplified reaction step.

Needs form (strand or two strands) according to the dna fragmentation after the amplification can be used one or both chimeric oligonucleotide primers in the method for the invention. Specifically, when the needs single stranded DNA, use a kind of chimeric oligonucleotide primer, and when the needs double-stranded DNA, use two kinds of primers.

The length of the chimeric oligonucleotide primer that uses in the method for the invention is not particularly limited, but preferred about 100 nucleotides of about 12-, about 40 nucleotides of 15-more preferably from about. Preferably the nucleotide sequence of described chimeric oligonucleotide and template nucleic acid are substantially complementary, so that it is annealed on the template nucleic acid under used reaction condition. Described primer 3 '-terminal or 3 '-end side is included in the sequence of the endonuclease identification of using in the step as described below.

Although for example purpose is not restriction the present invention, the oligonucleotides with structure that following general formula represents can be used as primer in DNA synthetic method of the present invention:

General formula: 5 '-dN_a-N _b-dN _c3 ' (a:11 or 11 above integers; B:0 or 1 or 1 above integer; C:0 or 1 or 1 above integer, prerequisite is that b and c can not be 0 simultaneously; DN: deoxyribonucleotide; N: unmodified ribonucleotide and/or modification ribonucleotide).

For example wherein a=11 or 11 above integers; And b=1 and c=0, b=2 and c=0, b=3-5 and c=0, perhaps the chimeric oligonucleotide primer that represents of the described general formula of b=2 and c=0-5 can be preferred for the present invention. 3 ' of the chimeric oligonucleotide primer that uses in the method for the invention-ribonucleotide length terminal or 3 '-end side is preferably the 1-matrix to the 15-matrix, more preferably the 1-matrix is to the 10-matrix, most preferably the 1-matrix is to the 5-matrix. C number in the general formula is not particularly limited, but can select to be used for any number of the inventive method. Common integer below preferred 5 or 5. C is 3,2 and 1 to be 4,3,2 can obtain better result than c respectively in reaction. Especially when c=0, can realize the most effective reaction.

Having wherein for chimeric oligonucleotide primer of the present invention, endonuclease cuts in the site that comprises ribonucleotide with the structure of archaeal dna polymerase from the DNA chain (primer extended chain) of primer extension. In other words, ribonucleotide is positioned on 3 ' of described chimeric oligonucleotide primer-end or the 3 ' end side, in order to cut with endonuclease. For example act on when being annealed to the double-stranded DNA that chimeric oligonucleotide primer extended DNA that template nucleic acid, described general formula represent produces as RNA enzyme H, cut described chimeric oligonucleotide primer at described ribonucleotide acid moieties. Then produce wherein at described Oligonucleolide primers and extend the double-stranded DNA that imports a breach between the synthetic DNA chain. Then carry out the strand replacement reaction of archaeal dna polymerase from gap portions. Yet, can use in the method for the invention any such chimeric oligonucleotide primer: can be used for extending nucleic acid chains, the cutting of available endonuclease and available archaeal dna polymerase from 3 ' of primer-end and realize strand replacement reaction.

For example the 394 type dna synthesizers of available Applied Biosystems Inc. (ABI) are according to the synthetic chimeric oligonucleotide primer with any nucleotide sequence of phosphoramidite method. Perhaps the synthetic described chimeric oligonucleotide primer of available any method comprises phosphotriester method, H-phosphate method and thiophosphate method.

(2) be used for endonuclease of the present invention

Below so any endonuclease can be used for the present invention: act on the double-stranded DNA that the chimeric oligonucleotide primer extended DNA described by above-mentioned (1) that is annealed to template nucleic acid produces, and the cutting extended chain is to realize strand replacement reaction. That is to say that described endonuclease is for partly producing the enzyme of a breach at the chimeric oligonucleotide primer of described double-stranded DNA. The example that can be used for endonuclease of the present invention includes but not limited to ribalgilase. Wherein can preferably use the endoribonuclease H (RNA enzyme H) of the RNA part of the double-strandednucleic acid that acts on DNA and RNA composition. Any ribalgilase with above-mentioned activity comprises and has a liking for warm endonuclease and heat-resisting endonuclease can be preferred for the present invention. For example colibacillary RNA enzyme H can be used for the about 50 ℃-Yue 70 ℃ reaction of the inventive method among the embodiment as described below. Also can preferably use commercially available thermostable rnase enzyme Hybridase^TMThermally-stabilised RNA enzyme H (Epicenter Technologies). In addition, described ribalgilase can be natural nucleus ribonuclease T. or variant. The RNA enzyme H enzyme unit that this paper indicates is the value according to the method representation of the described detection enzyme unit of reference example.

The endonuclease that uses in the methods of the invention such as the cleavage reaction efficient of RNA enzyme H may depend near the nucleotide sequence primer 3 '-end, and affect the amplification efficiency of needed DNA. So, must designing institute with the best primer of RNA enzyme H.

Term used herein " imports a breach " or " breach " refers to cut open in one of the two strands of double-strandednucleic acid inside. For example RNA enzyme H acts on DNA and the heteroduplex nucleic acid that the DNA that comprises ribonucleotide forms, and selectively cuts the chain that comprises ribonucleotide in the described two strands at the ribonucleotide position, therefore imports a breach in heteroduplex nucleic acid.

(3) be used for archaeal dna polymerase of the present invention

The archaeal dna polymerase that DNA is had the strand displacement activity can be used for the present invention. Specifically, can preferably use the archaeal dna polymerase of basic shortage 5 '-3 ' exonuclease activity.

Any archaeal dna polymerase with strand displacement activity all can be used for the present invention. The example comprises the archaeal dna polymerase variant of its 5 '-3 ' exonuclease activity of shortage of the Thermophilic Bacteria that derives from bacillus such as Bacillus caldotenax (after this being called B.ca) and bacillus stearothermophilus (after this being called B.st) and the large fragment (Klenow fragment) of colibacillary dna polymerase i. Have a liking for warm archaeal dna polymerase and hot resistant DNA polymerase and all can be preferred for the present invention.

B.ca is the about 70 ℃ Thermophilic Bacteria of optimum growth temperature. The Bca archaeal dna polymerase of known this bacterium has DNA dependent dna-polymerases activity, RNA dependent dna-polymerases active (reverse transcriptase activity), 5 '-3 ' exonuclease activity and 3 '-5 ' exonuclease activity.

Described enzyme can be for from the enzyme of its natural origin purifying or the recombinant protein that utilizes genetic engineering technology to produce. Utilize genetic engineering technology or other method that described enzyme is carried out such as the modification that replaces, lacks, adds or insert. The example of modification enzyme comprises Bca BEST archaeal dna polymerase (Takara Shuzo), and it is for lacking the Bca archaeal dna polymerase of its 5 '-3 ' exonuclease activity.

(4) being used for reaction buffer of the present invention forms

The reaction buffer that comprises buffer components, magnesium salts and various dNTP is used for the present invention. The example of the buffer components that can preferably use includes but not limited to Tricine, tris-hydrochloride and phosphate (sodium phosphate and potassium phosphate). The buffer solution that wherein comprises Tricine or phosphate buffered component can be preferred for the present invention. The final concentration scope of buffer components is 5-100mM, preferred 20-50 mM. The pH scope is 6.0-9.5, preferred 7.0-9.2. For example preferably use pH 7.5-9.2, the buffer solution that comprises 22-46mM Tricine or pH 7.0-8.0, comprise the buffer solution of 25-50mM potassium phosphate. The magnesium salts example that can preferably use includes but not limited to magnesium chloride, magnesium acetate or magnesium sulfate. The final concentration scope of magnesium salts is 1-20mM, preferred 2-10mM. Final concentration scope as the various dNTP (dATP, dCTP, dGTP and dTTP) of the substrate of DNA extension in the mixture is 0.1-3.0mM, preferred 0.2-1.2mM. The primer weight range that is used for 50 μ l reaction volumes is 1-1000pM, preferred 10-100pM. In addition, described reactant mixture can contain additive, in order to for example stablize amplified reaction. Can add final concentration and be 0.1% or lower BSA, final concentration be 10% or lower dimethyl sulfoxide (DMSO), final concentration be that 4mM or lower disalt sour amine or concentration are 0.01% or lower propylene diamine. Perhaps can contain NMP (1-methyl-2-pyrrolidine-diones), glycerine, PEG, dimethyl sulfoxide (DMSO) and/or formamide. Expection adds the non-specific annealing that such organic solvent can reduce Oligonucleolide primers.

In 50 μ l reaction volumes, be preferably 3-200U, more preferably 15-60U as the e. coli rna enzyme H content range of endonuclease example. In 50 μ l reaction volumes, be preferably 0.5-100U, more preferably 1-22U as the Bca BEST archaeal dna polymerase content range of DNA polymerase example. The preferred unit number of wherein said endonuclease is inferred with its type difference. In this case, the enzyme amount of regulating the component of buffer solution and needing to add is so that the amplified production amount is maximum. Under any circumstance, must optimize according to used enzyme type the component etc. of reaction buffer.

(5) the present invention is used for the method for amplification of nucleotide acid sequence

Utilize above-mentioned (1) described at least a Oligonucleolide primers and above-mentioned (2) described endonuclease and above-mentioned (3) described archaeal dna polymerase can carry out method of the present invention. The various dNTP (mixture of dATP, dCTP, dGTP, dTTP) that are used for the PCR method can the same substrate as described method extension with NTP. Described various dNTP can comprise the dNTP analog, and for example the 7-denitrification is mixed-dGTP, and prerequisite is that it is the substrate of used archaeal dna polymerase. Use chimeric oligonucleotide primer in the described method. For example adopt dna synthesizer can prepare described primer according to conventional synthetic method. Chimeric oligonucleotide primer and the conventional Oligonucleolide primers of combination can be used in the inventive method.

Can prepare or from any sample that may contain nucleic acid, separate the nucleic acid (DNA or RNA) that obtains to be used as in the methods of the invention template. The sample example that may contain nucleic acid includes but not limited to biological sample, whole blood for example, serum, buffy coat, urine, stool, cerebrospinal fluid, seminal fluid, saliva, tissue (for example cancerous tissue or lymph node) and cell culture (for example mammalian cell cultures or bacterial cell culture) contains the sample of nucleic acid such as viroid, virus, bacterium, fungi, yeast, plant and animal, suspects the sample (for example food or biologic product) of pollution or microbial infection (for example virus or bacterium) and may contain zoic sample such as soil and waste water. Described sample can be to contain the preparation of processing as mentioned above the nucleic acid of described sample acquisition according to known method. Can be used for formulation examples of the present invention and comprise that cytoclasis goods or classification separate the sample that the sample of nucleic acid in described goods, the described sample or specific nucleic acid molecule such as enrichment mRNA obtains. The nucleic acid that can preferably use the nucleic acid that adopts known method to increase to comprise in the described sample to obtain, for example DNA or RNA.

Adopt ad lib for example detergent cracking, ultrasonic processing, can prepare the preparation that comprises nucleic acid with bead vibration or stirring or French cell press from above-mentioned material. Preferably further process in some cases described preparation, with the described nucleic acid of purifying (for example in the situation that has the endogenous nuclease). In this class situation, utilize known method such as nucleic acid as described in phenol extraction, chromatography, ion-exchange, gel electrophoresis or the density gradient centrifugation purifying.

When the expectation amplification has the nucleic acid of the sequence that derives from RNA, can implement method of the present invention with the synthetic cDNA of reverse transcription reaction that utilizes described RNA to make template as template. People can all can be used for described method of the present invention for the preparation of any RNA of the primer of reverse transcription reaction, comprise the total RNA of RNA molecule such as sample, mRNA, tRNA and rRNA and specific RNA type.

Any primer that is annealed to template ribonucleic acid under used reaction condition all can be used for described reverse transcription reaction. Described primer can be to have and primer (Auele Specific Primer), oligomerization dT (deoxythymidine) primer of the nucleotide sequence of specific template RNA complementation and the primer (random primer) with random sequence. For specificity annealing, the length that is used for the primer of reverse transcription is preferably 6 or 6 above nucleotides, more preferably 9 or 9 above nucleotides. Consider the synthetic of oligonucleotides, described primer length is preferably 100 or 100 following nucleotides, more preferably 30 or 30 following nucleotides.

In addition, chimeric oligonucleotide primer can be used as the reverse transcription primer. Chimeric oligonucleotide primer also can be as the primer of the cDNA that utilizes reverse transcription to obtain as the strand replacement reaction in the method for the amplification of nucleotide acid sequence of the present invention of template. This primer can be any primer that has above-mentioned (1) described characteristic and can be used for the reverse transcription reaction of RNA.

Any enzyme with activity of utilizing the synthetic cDNA of RNA template all can be used for reverse transcription reaction. The example comprises the reverse transcriptase that various sources make, for example reverse transcriptase (AMV RTase), Moloney muroid leukemia virus reverse transcriptase (MMLV RTase) and the 2 type RAV reverse transcriptase (RAV-2 RTase) in avian monocytosis virus source. In addition, can use the archaeal dna polymerase that also has reverse transcriptase activity. The archaeal dna polymerase that at high temperature has the Thermophilic Bacteria of the enzyme of reverse transcriptase activity such as the archaeal dna polymerase of Thermus bacterium (for example Tth archaeal dna polymerase) and inferior spore Bacillus is preferred for the present invention. Although purpose is not restriction the present invention, the archaeal dna polymerase (Bca archaeal dna polymerase) of the archaeal dna polymerase of the Thermophilic Bacteria of preference such as inferior spore Bacillus such as the archaeal dna polymerase of B.st (Bst archaeal dna polymerase) and B.ca. For example the Bca archaeal dna polymerase does not need magnesium ion to reverse transcription reaction. And it can synthesize cDNA, the simultaneously secondary structure formation of inhibition template ribonucleic acid under hot conditions. The n DNA polymerase all can use with the variant with described enzyme of reverse transcriptase activity, and prerequisite is that they have described activity.

During with the inventive method amplification of nucleic acid, the amplification template nucleic acid purpose nucleic acid that can more effectively increase in advance. For example when nucleotide sequence that amplification exists in the micro-genomic DNA, at first increasing with suitable nucleic acid amplification method comprises the dna fragmentation of purpose nucleotide sequence. Then thus obtained amplification of DNA fragments is as template, to carry out amplification method of the present invention. Available method of the present invention is carried out first amplification step. Perhaps available known nucleic acid amplification method such as PCR method carry out first step. And can add specific nucleotide sequence at 5 '-end side of the primer that is used for described amplification step. When the fragment with this primer amplification was used as template, available chimeric oligonucleotide primer with the specific nucleotide sequence that adds above-mentioned primer carried out amplification method of the present invention. In other words, no matter need amplification section nucleotide sequence how, available common chimeric oligonucleotide primer carries out described nucleic acid amplification step by method of the present invention, wherein the dna fragmentation of pcr amplification is as template. Can unite the PCR that method of the present invention and utilization have the primer of the specific nucleotide sequence that is added in as mentioned above 5 '-end side and reach this purpose.

Usually people should prepare a pair of purpose nucleotide sequence Auele Specific Primer, so as in first nucleic acid amplification step the described sequence of specific amplification. Yet, application target nucleotide sequence Auele Specific Primer not, but and with random primer or a pair of primer amplification template nucleic acid that is selected from one group of ready-made degenerate primer of non-specific amplification nucleic acid fragment. Can reduce the primer of a plurality of template nucleic acids needs of amplification to number. 3778-3783 (1996)) or utilize the PCR method (DOP-PCR of the degenerate oligonucleotide guiding of the degenerate primer with flag sequence utilize the PCR method that for example adopts the random primer with flag sequence (Nucleic Acids Research, 24 (19):; Genomics, 13:718-725 (1992)) can reach and reduce the right purpose of primer. Each described primer has random sequence or degenerate sequence at 3 '-end. If the nucleic acid with the primer amplification with flag sequence carries out amplification method of the present invention as template, then can carry out method of the present invention with a kind of chimeric oligonucleotide primer. This chimeric oligonucleotide primer has the nucleotide sequence identical with described flag sequence. By adopting a kind of like this primer, use all nucleic acid of the primer amplification with same tag sequence all to can be used as template. Therefore by uniting method of the present invention and utilizing the nucleic acid amplification method of random primer or degenerate primer to supply in a large number various nucleotide sequences by low-down cost.

Utilize the template DNA chain again any archaeal dna polymerase of synthetic DNA chain all can be used for described nucleic acid amplification method. Such archaeal dna polymerase comprise pol I-type archaeal dna polymerase (for example e. coli dna polymerase I, Klenow fragment and Taq archaeal dna polymerase), α-type archaeal dna polymerase (for example archaeal dna polymerase of fierce hot-bulb bacterium, VENT archaeal dna polymerase, KOD archaeal dna polymerase and DEEP VENT archaeal dna polymerase) and non-α-, non--pol I-type archaeal dna polymerase (for example WO 97/24444 introduce archaeal dna polymerase). In addition, can preferably use mixture such as TaKaRa Ex Taq archaeal dna polymerase (Takara Shuzo) or the KOD dash archaeal dna polymerase (Toyobo) of at least two kinds of archaeal dna polymerases. And can preferably use following archaeal dna polymerase: for example the archaeal dna polymerase of the archaeal dna polymerase of B.ca, B.st, these archaeal dna polymerases lack the variant of its 5 '-3 ' exonuclease activity, 9 ° of N archaeal dna polymerases, Pfu (exo-) archaeal dna polymerase (Stratagene), Tth archaeal dna polymerase (Toyobo) and Tfl archaeal dna polymerases (Promega).

If in amplification method of the present invention, use linear DNA fragment (for example fragment of pcr amplification) as template, then import the sequence that is called compartment and can improve amplification efficiency. Compartment is positioned at 3 ' of template linear DNA fragment-end and is used between the 5 '-terminal annealing position of primer of the inventive method. For example preferred design is used for the primer of amplification method of the present invention, so that the length of compartment about 70 bases that are 1-ad lib, about 60 bases of 5-more preferably from about. The preferred bases radix of compartment can change with the primer sequence that is used for amplification method of the present invention. Can determine the optimal spacing part with reference to the disclosed content of the embodiment of the invention. For example use in advance fragment of pcr amplification, so that compartment joins 3 ' of amplimer annealing section of the present invention, such amplified fragments can be used as the template of amplification method of the present invention. In one embodiment, use in advance a kind of primer amplification template nucleic acid. Such primer has a section of another primer of section of a section of compartment, amplimer of the present invention and amplification of nucleic acid in 5 '-3 ' direction. So the fragment of amplification is then as the template in the amplification method of the present invention. The section that is used for another primer of amplification of nucleic acid can be any section for the primer of nucleic acid amplification method such as PCR method. The section that perhaps is used for another primer of amplification of nucleic acid can be a section of another amplimer of the present invention.

Double-stranded DNA all can be used as template DNA such as the genomic DNA that separates or PCR fragment and single stranded DNA such as total RNA or mRNA in the present invention through the cDNA that reverse transcription reaction makes. The preferred single stranded DNA that uses the double-stranded DNA sex change to become.

If linear dsdna such as pcr amplification product then can be removed the denaturing step in the amplification method of the present invention as template. Make the annealing position of primer of the present invention be positioned at terminal inboard about 50 the base places of described DNA and can remove denaturing step. If the nucleic acid that needs amplification to have the RNA sequence then can utilize RNA as the reverse transcription reaction of template and utilize the cDNA of described reverse transcription reaction generation as the dna amplification reaction of template with a kind of archaeal dna polymerase in DNA synthetic method of the present invention. Described archaeal dna polymerase has reverse transcriptase activity and strand displacement is active.

The appropriate length of described template is for because exist enough parts of target sequence in complete target sequence or the described at least fragment to provide well-bound length for primer sequence.

If dna profiling is double-stranded DNA, then making in the methods of the invention described DNA sex change is single stranded DNA, so that primer can be attached on the template DNA chain. Double-stranded DNA is remained under the denaturation temperature (for example about 95 ℃). Other method comprises the method for wherein utilizing pH to raise. Oligonucleolide primers in this case, should reduce pH, in order to can be attached on the target of amplified reaction. If after the double-stranded DNA sex change is single stranded DNA or RNA when the template, utilize reverse transcription reaction to prepare cDNA (single stranded DNA) after, continuous amplification of nucleotide acid sequence under constant temperature.

" continuously " refers to form reaction temperature or reactant mixture, and variation ground reacts. " constant temperature " used herein refers to the substantially invariable condition of temperature, and the enzyme of each step and nucleic acid chains have function under this temperature.

Under bound by theory not, consider in the method for amplification of nucleotide acid sequence of the present invention continuously, repeat parallel carrying out (for example under constant temperature) following steps:

[1] make template DNA be annealed to step at least a Oligonucleolide primers;

[2] realize from the step of 3 ' of described primer-end extension with the reaction of the DNA of described template DNA complementation;

[3] step of the DNA chain of usefulness endonuclease cutting step [2] extension;

[4] realize from the reaction of the 3 '-terminal extended DNA in the described site of step [3] cutting, simultaneously release steps [2] extended DNA chain and can not make it from the step of template DNA degraded; And

[5] with the double stranded polynucleotide repeating step [3] of step [4] acquisition and the step of step [4].

Can use have a liking for warm archaeal dna polymerase for example the Klenow fragment under typical temperature (for example 37 ℃), carry out above-mentioned reaction. Adopt thermostable enzyme (endonuclease and archaeal dna polymerase) also can under high temperature (for example 50 ℃ or higher, or 60 ℃ or higher), carry out. In this case, non-specific primer annealing is suppressed, so that the specificity of DNA cloning increases. And solved the problem that template DNA forms secondary structure, so that the extension ability of archaeal dna polymerase improves. In one embodiment, can in described method, carry out continuously the amplification of reverse transcription reaction and nucleotide sequence. In this case, archaeal dna polymerase with reverse transcriptase activity of use in conjunction reverse transcriptase and above-mentioned reaction or utilization can increase and have the DNA of the sequence that RNA produces.

A first aspect of the present invention is that a kind of single stranded DNA that adopts is as the method for template and at least a chimeric oligonucleotide primer amplification of nucleotide acid sequence.

It is a kind of method of amplification of nucleotide acid sequence, it is characterized in that described method comprises:

(a) with the part of the nucleotide sequence of at least a and described nucleic acid basic complementary primer and archaeal dna polymerase processing template nucleic acid, with primer extended chain synthetic and the template complementation, wherein said primer is the chimeric oligonucleotide primer that comprises deoxyribonucleotide and ribonucleotide, described ribonucleotide is positioned on 3 '-end side of 3 ' of primer-end or primer, in order to cut with endonuclease;

(b) with the primer extended chain of described endonuclease at the double-strandednucleic acid of site cutting step (a) acquisition that comprises described ribonucleotide; And

(c) use the archaeal dna polymerase with strand displacement activity, from primer 3 '-terminal extension partly and the nucleotide sequence of template complementation of described double-strandednucleic acid, wherein the primer extended chain of cutting step (b) acquisition reaches strand displacement.

A second aspect of the present invention is a kind of with the method for single stranded DNA as template and at least two kinds of chimeric oligonucleotide primer amplification of nucleotide acid sequences of the present invention.

It is the method for at least two kinds of primer amplification nucleotide sequences of a kind of application, it is characterized in that described method comprises:

(b) with the primer extended chain of described endonuclease at the double-strandednucleic acid of site cutting step (a) acquisition that comprises described ribonucleotide;

(c) with the archaeal dna polymerase with strand displacement activity, primer 3 '-terminal extension partly and the nucleotide sequence of template complementation from described double-strandednucleic acid, wherein the primer extended chain of cutting step (b) acquisition reaches strand displacement, and wherein step (b) is reused the double-strandednucleic acid that contains the primer extended chain that regenerates;

(d) with at least a primer of step (a) use and the release displacement chain template of archaeal dna polymerase treatment step (c) acquisition of being different from, with primer the extended chain synthetic and complementation of displacement chain, wherein be different from the primer that step (a) uses and be chimeric oligonucleotide primer, this primer is with the basic complementation of the part of the nucleotide sequence of displacement chain and contain deoxyribonucleotide and ribonucleotide, described ribonucleotide is positioned on 3 '-end side of 3 ' of primer-end or primer, in order to cut with endonuclease;

(e) with the primer extended chain of endonuclease at the double-strandednucleic acid of site cutting step (d) acquisition that contains ribonucleotide; And

(f) with the archaeal dna polymerase with strand displacement activity, primer 3 '-terminal extension partly and the nucleotide sequence of template complementation from described double-strandednucleic acid, wherein the primer extended chain of cutting step (e) acquisition reaches strand displacement, and wherein step (e) is reused the double-strandednucleic acid that contains the primer extended chain that regenerates.

The present invention the 3rd or fourth aspect be a kind of single stranded DNA that after the pre-treatment step of double-stranded DNA sex change for single stranded DNA, obtains that wherein utilizes as template, amplification is according to the method for the nucleotide sequence of the present invention first or second aspect.

The the of the present invention the 5th or the 6th aspect is that a kind of RNA that wherein utilizes prepares the cDNA that obtains after the strand cDNA step as template as template through reverse transcription reaction, and amplification is according to described first or the method for the nucleotide sequence of second aspect.

" the primer extended chain that regenerates " used herein refer to from because strand displacement be reused for that the Oligonucleolide primers that copies extends, with the DNA chain of template nucleotide sequence complementation.

" reusing " used herein refers to again utilize the double-stranded DNA that is comprised of template nucleotide sequence and the primer extended chain that regenerates in the strand displacement step.

The present invention each above-mentioned aspect in, at first be annealed on the described DNA with the chimeric oligonucleotide primer of template single stranded DNA complementation. Then utilize effect all the other sequences extensions from 3 ' of described primer-end along described template DNA and the DNA (primer extended chain) of described template DNA complementation of archaeal dna polymerase, with synthetic dsdna. A kind of endonuclease acts on described double-stranded DNA, in a site of 3 '-end side of the ribonucleotide acid moieties of described chimeric oligonucleotide primer it is cut. Described endonuclease can not cut other position of described DNA. Therefore, endonuclease plays to import at described double-stranded DNA the breach enzyme of a breach. Although the present invention is not bound by theory, described endonuclease may change the structure of the double-stranded DNA of described chimeric oligonucleotide primer and template DNA composition. 3 '-end that archaeal dna polymerase with strand displacement activity imports breach from described double-stranded DNA is the extended DNA chain again, produces new primer extended chain, discharges simultaneously the DNA in described breach 3 '-terminal downstream. Therefore, the new previous synthetic primer extended chain of primer extended chain displacement.

Available two kinds of primers carry out the method for amplification of nucleotide acid sequence of the present invention, described primer namely with the chimeric oligonucleotide primer of template nucleic acid complementation and the chimeric oligonucleotide primer another kind of and complementation of displacement chain. In this case, a kind of primer is attached to the template DNA chain and causes strand replacement reaction, and another kind of primer is attached to the displacement chain that discharges because of described strand replacement reaction, and begins another strand replacement reaction. If obviously utilize this on the one hand, then a kind of product of primer can be used as the template of another primer. Therefore, because the increase of template amount, so the amplified production amount increases with nonlinear way.

When carrying out the method for amplification of nucleotide acid sequence of the present invention with double-stranded DNA as template, before or after described double-stranded DNA sex change, in reactant mixture, add a kind of chimeric oligonucleotide primer, 4 kinds of triphosphate deoxyribose nucleotides (dNTP), a kind of archaeal dna polymerase and a kind of endonuclease. Do not use thermostable enzyme if utilize the described double-stranded DNA of heat treatment sex change, then preferably after sex change, add described enzyme.

As described in above-mentioned (2), select to be used for the endonuclease of described method, so that it is in the ribonucleotide acid moieties cutting chain of primer. Preferably cut described chain in 3 ' site of described ribonucleotide. People should select archaeal dna polymerase, so that it is with the suitable speed breach DNA chain that dissociates.

Be used for archaeal dna polymerase of the present invention and synthesize downstream extended chain from the breach site, replace simultaneously previous extended DNA chain. Importantly, described archaeal dna polymerase does not have 5 '-3 ' exonuclease activity, otherwise the described displacement chain of its degradable. For example Klenow fragment (the exonuclease deficient variant of e. coli dna polymerase I), the similar fragment (New Enland Biolabs) that obtains from the Bst archaeal dna polymerase and the Bca BEST archaeal dna polymerase (Takara Shuzo) of B.ca can be used as such archaeal dna polymerase. Also can use Gene, the Sequenase 1.0 described in the 97:13-19 (1991) and Sequenase 2.0 (United States Biochemical) and T5DNA polymerase and 29 archaeal dna polymerases. Can suppress described activity if add suitable inhibitor, the polymerase that then usually has exonuclease activity can be used for DNA synthetic method of the present invention.

Can under different temperatures, carry out the method for amplification of nucleotide acid sequence of the present invention or can carry out by constant temperature. Variations in temperature refers to that the reaction temperature of corresponding steps changes, but described variation can not disturbed the reaction of each step. Specifically, variations in temperature refer to variations in temperature to the synthetic reaction of the annealing that is suitable for each primer for example, complementary strand, make complementary strand produce breach and strand replacement reaction.

On the other hand, constant temperature refers to that constant and each step of the reaction temperature of each step carries out under substantially invariable temperature. Must select temperature to optimize the reaction condition of two kinds of situations.

One of feature of the method for amplification of nucleotide acid sequence of the present invention is that described method does not need the up-down adjustment temperature at nucleic acid between synthesis phase. Therefore, the invention provides the method for constant temperature synthesizing ribonucleotide sequence. Many conventional nucleic acid amplification methods need the up-down adjustment temperature, so that target chain and synthetic chain dissociate. For this reason, these methods need for example thermal cycler of special consersion unit. Yet, only use to keep the equipment of constant temperature just can carry out method of the present invention.

As mentioned above, can under single temperature, carry out method of the present invention. Preferably carry out under the following conditions: select reaction temperature and stringent condition level that the non-specific annealing of primer is reduced, and make described primer specificity be annealed to the template nucleotide sequence. Although purpose is not restriction the present invention, method of the present invention can be utilized thermostable enzyme and at high temperature carry out as mentioned above. In addition, be preferably under the suitable temperature of the used enzymatic activity of abundant maintenance and carry out method of the present invention, in order to keep high-caliber reaction efficiency. Difference is preferably about 20 ℃-Yue 80 ℃, more preferably from about 30 ℃-Yue 75 ℃, most preferably from about 50 ℃-Yue 70 ℃ although reaction temperature is with used enzyme. Especially when under hot conditions, carrying out described reaction, preferably use the primer longer than the primer length that reacts under the normal temperature. For example can determine to be fit to primer sequence and the length of described reaction temperature with reference to its Tm value. On the other hand, can use the commercial software for the design primer, for example OLIGOTM primer analysis software (Takara Shuzo). For example when reaction temperature is used 55 ℃-60 ℃ or 65 ℃, the primer that is used for the inventive method can be for example to be ad lib 12-100 length of nucleotides, preferably 14-50 length of nucleotides, more preferably 15-40 length of nucleotides. One of effect that the rising reaction temperature produces is the problem that has solved template DNA formation secondary structure. The rising reaction temperature makes it possible to the nucleic acid of needs that increases, even use the high nucleic acid of GC content also like this as template. An and district of same effectively amplification long-chain length. In the about 20kbp of about 100 bp-, the about 4.3kbp of especially about 200bp-, be more particularly the about 1500 bp scopes of about 250bp-and observe such effect.

Use in the methods of the invention to have the archaeal dna polymerase (for example Bca BEST archaeal dna polymerase) of reverse transcriptase activity so that can routine be carried out by RNA amplification of nucleotide acid sequence, comprising the step (reverse transcription reaction) for preparing cDNA with RNA. Perhaps, independently carrying out preparing the product that the step of cDNA obtains by RNA is that cDNA can be used as template DNA in the methods of the invention.

In all cases, repeat the reaction of the inventive method, until with suitable method for example fermentoid or reduce reaction temperature and end till, perhaps until react and run out of wherein till a kind of reagent.

Fig. 1 illustrates an embodiment, wherein uses single stranded DNA as template and two kinds of primers. The capable corresponding steps of carrying out of continuous strand is as described below:

(1) make the template single stranded DNA be annealed to the step of chimeric oligonucleotide primer;

(2) realize that reaction from 3 ' of described primer-terminal extended DNA is to form the step of primer extended chain;

(3) comprise the step of the site cutting of ribonucleotide at primer with endonuclease;

(4) with the step of archaeal dna polymerase from the cleavage site realization strand displacement of step (3);

(5) reuse the step of the double-stranded DNA that primer extended chain that the template that obtained by step (4) and step (3) regenerate forms, simultaneously the displacement chain of use release in step (6) reaction and following steps;

(6) make the Oligonucleolide primers that is different from step (1) be annealed to the step of the release displacement chain of step (5) as template;

(7) realize that by 3 ' of described primer-end the DNA extension is to form the step of primer extension chain;

(8) carry out step of cutting with endonuclease in the site that primer comprises ribonucleotide;

(9) with the step of archaeal dna polymerase from the cleavage site realization strand displacement of step (8); And

(10) reuse the template of step (9) acquisition and the step of the primer extension chain that step (8) produces again.

When double-stranded DNA was used as template, each single stranded DNA that obtains after the described double-stranded DNA sex change was as the template of step (1).So the amplified production amount is more than obtaining the amplified production amount with single stranded DNA as template.In addition, compare with the time of needed detection amplified production when single stranded DNA is used as template, the time of detecting amplified production is shorter.

The inventive method that is used for the amplification of nucleotide acid sequence can be used for the experimental technique of various application nucleotide sequences amplifications, comprises detection, mark and nucleic acid is checked order.

In addition, the polynucleotide TRAP that the inventive method that is used for the amplification of nucleotide acid sequence can be used for the in-situ nucleic acid TRAP, the method for amplification of nucleic acid such as DNA chip or wherein a plurality of district increase simultaneously on solid support.

One of the feature that is used for the inventive method of amplification of nucleotide acid sequence can prepare single stranded DNA for it.For this reason, one or both chimeric oligonucleotide primers can be used for described method.If for example use two kinds of Oligonucleolide primers, then can use the primer ratio that is used for so-called asymmetric PCR method and carry out method of the present invention, wherein use excessive a kind of Oligonucleolide primers to carry out amplified reaction with respect to another kind of Oligonucleolide primers amount.Therefore, with respect to another chain product amount, the substitution product amount of a chain is excessive.

According to the inventive method that is used for the amplification of nucleotide acid sequence, can prepare the single stranded DNA that is substantially free of its complementary strand.For example can easily produce the single stranded DNA that is used to prepare the material that contains fixed nucleic acid such as DNA chip in a short period of time, be used to the big primer that detects the ssDNA probe of target nucleic acid or be used for long-chain PCR method.By using the alternative only amplification of method of the present invention adopted sequence or antisense sequences are arranged.Therefore, the present invention can effectively have the method for the nucleic acid that adopted sequence or antisense sequences are arranged as preparation.

In addition, the inventive method that is used for the amplification of nucleotide acid sequence does not need to use the conversion unit of attemperation in time.Therefore, can carry out amplified reaction with high volumetric reaction mixture.So, suitability for industrialized production nucleic acid (for example medicinal use nucleic acid) in a large number.

The service efficiency of primer that is used for the inventive method of amplification of nucleotide acid sequence is about 100%, it may than ordinary method such as the high 5-10 of PCR method doubly more than.

(6) be used for the test kit of the inventive method of amplification of nucleotide acid sequence

The invention provides and be used for the test kit of the method for the amplification of nucleotide acid sequence of first to the 6th aspect as mentioned above.In one embodiment, described test kit is a packaged form, wherein comprises about use the specification sheets of archaeal dna polymerase and endonuclease in strand replacement reaction.Equally, comprise the test kit that has the active archaeal dna polymerase of strand displacement, endonuclease and be used for the buffer reagent of strand replacement reaction and be preferred for method of the present invention.On the other hand, can select to use commercially available have active archaeal dna polymerase of strand displacement and/or endonuclease according to specification sheets.In addition, described test kit can comprise the reagent that is used for reverse transcription reaction that uses when RNA is used as template.As described in above (3), can from various archaeal dna polymerases, select to be used for archaeal dna polymerase of the present invention.As described in above (2), can from various endonucleases, select endonuclease.Damping fluid with above (4) described reaction buffer component is preferably used as the damping fluid of strand replacement reaction.

" specification sheets " is printing material, introduces the method for using described test kit, and for example preparation is used for the compositions and methods of strand replacement reaction, the reaction conditions of recommendation etc.Specification sheets comprises brochure or the loose service manual of page or leaf form, the explanation that is pasted on the label on the test kit and the surface of package of test kit is housed.The information that electronic media such as Internet disclose or provide also is provided specification sheets.

(7) be used to the test kit that detects the method for nucleotide sequence of the present invention and be used for described method

Utilization is used for the target nucleic acid that the inventive method of amplification of nucleotide acid sequence can test sample.Detection method comprises:

(a) with the inventive method amplifying target nucleic acid of aforesaid amplification of nucleotide acid sequence; And

(b) detect the target nucleic acid that above step increases.

Described method can be used for detecting or the quantitative specific gene in the sample.In other words, can detect or quantitatively the specific gene in the sample that contains nucleic acid such as DNA or RNA with suspicion.Can detect or quantitatively the sample example of specific gene include but not limited to biological sample, for example whole blood, serum, buffy coat, urine, ight soil, cerebrospinal fluid, seminal fluid, saliva, tissue (for example cancerous tissue or lymphoglandula) and cell culture (for example mammalian cell cultures or bacterial cell culture), the sample that contains nucleic acid such as viroid, virus, bacterium, fungi, yeast, plant and animal, suspect the sample (for example food or biotechnological formulation) of pollution or microbial infection such as virus or bacterium, and may contain biological sample such as soil and waste water.For example according to whether having viroid, virus, fungi, bacterium or other target microorganism deutero-specific gene or its content, can detect or quantitative viroid, virus, fungi, bacterium or other microorganism in the sample.And method of the present invention can be used for distinguishing biological genotype or detect the expression of gene level.In described detection method, can preferably use RNA and DNA the two as template nucleic acid.

The currently known methods that is used to detect nucleic acid can be used for step (b).The example of these class methods comprises the hybridization detection that utilizes electrophoresis detection to have the reaction product of specific size and utilize probe.Fluorescent substance such as ethidium bromide are used for electrophoresis detection.Utilize the hybridization of probe to join together with electrophoresis detection.Available radio isotope or non-radioactive substance such as vitamin H or fluorescent substance label probe.In addition, have at step (a) applying marking Nucleotide and help detect amplified production.Fluorescence polarization method, fluorescent energy transition etc. also can be used for described detection.Can automatization detect target nucleic acid or pass through to make up suitable detection system quantifying target nucleic acid.

Can be used for detection method of the present invention with fluorescent substance more than 2 kinds or 2 kinds with ribonucleotide (RNA) probe of the range mark that produces the quencher state.Probe is emitting fluorescence not.When probe is annealed to by with the DNA of probe complementary target nucleic acid amplification the time, RNA enzyme H digests probe.Then increase the distance between the fluorescent substance on the described probe, make emitting fluorescence.Therefore, emitting fluorescence represents to exist target nucleic acid.If RNA enzyme H is used for the inventive method of amplification of nucleotide acid sequence, then can in reaction mixture, only adds probe and just can detect target nucleic acid.For example make up fluorescent substance 6-Fluoresceincarboxylic acid (6-FAM) and N, N, N ', N '-tetramethyl--6-carboxyl rhodamine (TAMRA) can be preferred for label probe.

The inventive method of amplification of nucleotide acid sequence does not need use equipment such as thermal cycler under constant temperature.The primer number that is used for amplification method of the present invention also can be 1 or 2, and the primer number that uses than ordinary method lacks.Can be used in the inventive method because be used for reagent such as the dNTP of PCR etc.,, can reduce usage charges so compare with ordinary method.Therefore, the inventive method can be preferred for the wherein conventional field of detecting, and comprises genetic experiment.Compare with the PCR method, the inventive method provides more substantial amplified production with the short time.So the inventive method can be used as easy, the quick and responsive method that detects gene.

The present invention further provides the test kit of the method that is used to detect target nucleic acid.As mentioned above, the test kit that is used for the inventive method of amplification of nucleotide acid sequence can be used as this test kit.Reagent such as probe that test kit can further comprise the chimeric oligonucleotide primer that is used for amplifying target nucleic acid and be used to detect the target nucleic acid of amplification.

(8) contain the material of the present invention and the production method thereof of fixed nucleic acid array in prospective region

DNA chip (being also referred to as dna microarray or DNA array) is the material with fixed nucleic acid, and wherein the various arrangement of fragments of gene or DNA are fixed on prospective region or predetermined position on solid support such as the slide glass.The DNA chip is used for detecting nucleic acid samples and whether has the nucleic acid that has with the array fixed dna complementary sequence of DNA chip prospective region.The following detection: make the DNA chip with the nucleic acid sample of specimen preparation, be preferably the labeling nucleic acid sample and contact and hybridize.Because the DNA chip can detect or the quantitative nucleic acid quantity in the sample with step, so it is unusual useful method, it has promoted the analysis of genetic expression or the analysis of sudden change or polymorphism greatly.Wherein arranging fixedly in prospective region, the DNA chip of double-strandednucleic acid makes it through being used for hybridization after the suitable sex change.Wherein arrange fixing DNA chip and be particularly preferred for detecting target nucleic acid with the target nucleic acid complementary single stranded DNA that needs to detect in prospective region.

As mentioned above, method of the present invention can single stranded form the DNA that needs of amplification.Although can use any method of purifying amplified production, preferably utilize the purifying of isopropanol precipitating.Thus obtained DNA is preferably the single stranded DNA that is substantially free of its complementary strand, can be preferably used as the dna fragmentation that is fixed on the DNA chip.Therefore, method of the present invention is preferably used as the method that preparation need be arranged the DNA that is fixed on prospective region, to produce the DNA chip.Any insoluble upholder all can be used as the upholder of arranging fixing thus obtained DNA on its prospective region, but the film shape upholder that plate shaped upholder that preferred use glass or plastics are made and nitrocellulose or nylon are made.The currently known methods of fixed nucleic acid can be used for described fixing.DNA can directly be fixed on the upholder.Perhaps, DNA is fixed on the upholder after can or connecting a plurality of dna moleculars by a suitable joint.

Can detect or quantitatively with the material (for example DNA chip) with fixed nucleic acid on the target nucleic acid of nucleic acid hybridization, the DNA arrangement of the inventive method amplification on described material is fixed on the prospective region.Can followingly finish this detection or quantitatively: described material is contacted, with the nucleic acid sample that contains the specimen preparation of target nucleic acid with suspection to hybridize.Wherein, the single stranded DNA of method of the present invention amplification is arranged the DNA chip that is fixed in the prospective region, compares with conventional material, make can with more convenient operation, more hypersensitivity and also more high duplication detect target nucleic acid.

(9) be used for the inventive method of mass production nucleic acid

As mentioned above, one aspect of the present invention is provided for the method for amplification of nucleotide acid sequence, and this method can be carried out under constant temperature.Can the described method of following usefulness produce the nucleic acid that needs: mix the template nucleic acid of the nucleic acid that needs amplification and the various compositions of reaction needed, and described mixture is reacted under constant temperature.Because PCR method need change the temperature of reaction mixture in time, thus its reaction volume be restricted to wherein can controlled temperature volume (it is following to be generally 200 μ l or 200 μ l).Therefore, be difficult to scale up volume.On the contrary, there is not such restriction in method of the present invention.Can produce a large amount of nucleic acid by increasing the reaction mixture volume.In the method for the invention, by the synthetic countless complementary strand molecules of a template molecule.And utilize these complementary strand molecules can nucleic acid as template.Thereby, by suitable selection template and the primer nucleic acid that needs of mass production effectively.In addition, different with PCR method is, method of the present invention does not need special equipment or complicated temperature variation, and such fact makes it have advantage aspect equipment cost and the cost of energy.So the inventive method is the good commercial run of mass production nucleic acid.

In addition, the inventive method can be used as the method for a large amount of supply dna fragmentations, for example needs to be fixed on the supply of a large amount of dna fragmentations on the DNA chip.Specifically, in one embodiment, can obtain dna fragmentation in a large number with easy reactions steps.In another embodiment, can use limited amount primer to obtain various dna fragmentations.Use known nucleic acid amplification method (for example PCR method) amplification can add in one embodiment of back in advance as the step of the nucleic acid of the template in the inventive method.3778-3783 (1996)) or use the PCR (DOP-PCR of the degenerate oligonucleotide guiding of degenerated primer the method (Nucleic Acids Research, 24 (19): that for example have the random primer amplification of nucleic acid of flag sequence according to utilization; Genomics, 13:718-725 (1992)), can use the various nucleic acid of primer amplification of limited quantity as template.For above-mentioned steps produce as for all nucleic acid of template, use one or more primers just can carry out amplification method of the present invention.Can be as being issued to this purpose: be designed for the primer of amplification method of the present invention, so that it is equivalent to be added in the flag sequence of random primer or degenerated primer.Therefore, compare with ordinary method, combined preparation can be in a large number as the appropriate steps of the nucleic acid of template and the inventive method and be low-costly supplied various dna fragmentations.

The medicinal compositions that comprises nucleic acid can contain double-stranded DNA of expressing useful polypeptide in cell or the strand antisense DNA that suppresses destination gene expression.Use suitable method for example the carrier of transgenosis (for example liposome) described nucleic acid is sent in the organism.The inventive method that is used to produce nucleic acid is preferred for producing the single-chain nucleic acid or the double-strandednucleic acid of a large amount of medicinal uses.In addition, utilize the inventive method to produce at an easy rate to contain the nucleic acid of the dNTP analogue that for example suppresses the nucleic acid in vivo degraded.

Because the dna fragmentation of the present invention's amplification is made up of common Nucleotide, so utilize the restriction enzyme sites among the described DNA DNA subclone that is increased can be gone into suitable carrier.In addition, for example available restriction enzyme treatment DNA that does not have the RFLP problem.Therefore, described DNA can be widely used in the genetic experiment field.Because the dna fragmentation of the present invention's amplification is made up of normal oligodeoxynucleotide, so the promoter sequence of RNA polymerase can add in the fragment that is increased.The fragment that is increased can be used as the template of synthetic RNA, and described RNA for example can be used as probe.Certainly, use fluorescently-labeled dNTP and replace normal dNTP to carry out the inventive method of amplification of nucleotide acid sequence, can produce fluorescently-labeled dna probe.

Because the fragment of the final amplification of the inventive method is not at two ends and the primer complementary nucleotide sequence that is used to increase, so can reduce because the pollution due to the leaving over of amplified production.Therefore, the inventive method can be used for the genetic experiment of wherein conventional amplification same sector etc.

The feature that is used for the inventive method of amplification of nucleotide acid sequence is listed below:

1. it can be by a large amount of nucleic acid of a small amount of template amplification.When using two kinds of primers, amplified production increases with quadratic power.

2. but its constant temperature carries out.In this case, it does not need to use the equipment such as thermal cycler.Therefore, reaction volume can easily amplify.

3. adopt one or both chimeric oligonucleotide primers and two kinds of enzymes (archaeal dna polymerase and endonuclease) to carry out amplified reaction usually.

4. because by the synthetic countless DNA chains of a primer molecule, so the primer amount can not limit the amplified production amount.And the primer utilising efficiency is about 100%, and it is more much higher than the primer utilising efficiency of PCR method.

5. according to purpose alternative amplification strand or double-stranded DNA.

6. because it need be such as (α-S) the dNTP analogue of dNTP is used for amplified reaction, so reagent cost is cheap.And can obtain not contain the natural form nucleic acid of dNTP analogue.

7. it can be by associating the inventive method and another kind of nucleic acid amplification method and low cost, supply the dna fragmentation that increases in a large number.

As mentioned above, the inventive method is fit to industrial-scale production nucleic acid.

Embodiment

Following examples are illustrated the present invention in more detail, and can not be construed as limiting the invention scope.

Reference example

Detect the unit value of the RNA enzyme H that uses in the methods of the invention in accordance with the following methods.

(1) the used reagent solution of preparation

Be used for measuring active reaction mixture: sterilized water contains the following material of described final concentration: 40mM tris-hydrochloride (7.7,37 ℃ of pH), 4mM magnesium chloride, 1mM DTT, 0.003%BSA, 4% glycerine and 24 μ M poly (dT).

Poly-[8- ³H] poly-[8-of adenylic acid (AMP) solution: 370kBq ³H] adenylic acid (AMP) solution is dissolved in the 200 μ l sterilized waters.

Polyadenylic acid solution: making the polyadenylic acid dilution with aseptic ultrapure water is 3mM concentration.

Enzyme diluting soln: the material that contains the following stated final concentration in the sterilized water: 25mM tris-hydrochloride (7.5,37 ℃ of pH), 5mM 2 mercapto ethanol, 0.5mM EDTA (7.5,37 ℃ of pH), 30mM sodium-chlor and 50% glycerine.

The calf thymus DNA of preparation thermally denature: the 200mg calf thymus DNA is suspended expand in 100ml TE damping fluid.Being pursuant to the absorbancy that UV 260nm detects, is 1mg/ml concentration with aseptic ultrafiltration water with described solution dilution.Make diluting soln in 100 ℃ of heating 10 minutes, cooling fast in ice bath then.

(2) be used for the method for detection of active

In 985 μ l reaction mixtures, add the poly-[8-of 7 μ l ³H] adenylic acid (AMP) solution, the activity of above-mentioned to detect (1) preparation solution.In 37 ℃ of incubation mixtures 10 minutes.Adding 8 μ l polyadenylic acids in mixture, to make its final concentration be 24 μ M.Mixture is again in 37 ℃ of incubations 5 minutes.Make the poly-[8-of 1000 μ l thus ³H] rA-is poly--the dT reaction mixture.Make 200 μ l reaction mixtures in 30 ℃ of incubations 5 minutes then.The enzyme solution that wherein adds the suitable serial dilution degree of 1 μ l.Different time takes out 50 each sample of μ l and is used for subsequently detection from reaction mixture.Add enzyme to time of sampling (minute) be defined as Y.Adding 1 μ l enzyme diluting soln replaces enzyme solution and prepares 50 μ l as the reaction mixture of measuring total CPM or blank.The calf thymus DNA solution and 300 μ l, 10% trichoroacetic acid(TCA) (300 μ l ultrapure waters are used to detect total CPM) that in described sample, add 100 μ l100mM trisodium phosphates, 50 μ l thermally denatures.Make mixture in 0 ℃ of incubation 5 minutes, centrifugal 10 minutes then with 10000rpm.After centrifugal, the supernatant liquor of getting 250 μ l generation places bottle.Wherein add 10ml Aquasol-2 (NEN Life Science Products).Detect CPM with the liquid flashing counting device.

(3) Units of Account

Calculate the unit value of every kind of enzyme according to following formula:

Unit/ml={ (the blank CPM of the CPM-that records) * 1.2 ^** 20 * 1000 * extent of dilution) } 200 (μ l)/(total CPM * Y (minute) * 50 (μ l) * 9 ^*)

1.2 ^*: the poly-[8-that contains among total CPM of per 50 μ l ³H] rA-is poly--and dT measures (nM).

9 ^*: correction coefficient.

Embodiment 1

(1) synthetic template DNA and primer

Utilize synthetic single stranded DNA with 99 bases and the primer that uses in the present embodiment of dna synthesizer (Applied Biosystems) as template.The nucleotides sequence of the single stranded DNA of 99 bases is shown in the SEQ ID NO:1 of sequence table.The basic nucleotide sequence of upstream primer and downstream primer is shown in the SEQ ID NO:2 and 3 of sequence table respectively.Below introduce the structure of present embodiment the primer in detail:

Primer is to 1: the combination of primers that has nucleotide sequence shown in the SEQ ID NO:2 or 3 of sequence table and all be made up of deoxyribonucleotide;

Primer is to 2: wherein primer to each primer of 1 in, 3 '-terminal first and second deoxyribonucleotide replace by ribonucleotide and 3 '-combination of primers that the phosphate bond of 5 '-end side of terminal second ribonucleotide is replaced by a thiophosphoric acid key;

Primer is to 3: wherein primer to each primer of 1 in, the combination of primers that 3 '-terminal deoxyribonucleotide is replaced by ribonucleotide and the phosphate bond of 5 '-end side of this ribonucleotide is replaced by the thiophosphoric acid key;

Primer is to 4: wherein primer to each primer of 1 in, 3 '-terminal first and second combination of primers that deoxyribonucleotide is replaced by ribonucleotide; And

Primer is to 5: wherein primer to each primer of 1 in, 3 '-terminal the 3rd and the 4th deoxyribonucleotide replaced by ribonucleotide and the phosphate bond of the 4th the ribonucleotide 5 '-end side of 3 '-end is replaced by the thiophosphoric acid key combination of primers.

(2) amplified reaction

Use Bca BEST archaeal dna polymerase (Takara Shuzo) and clone's ribonuclease H (Takara Shuzo) to detect the following 1-7 model reaction system of introducing, wherein Bca BEST archaeal dna polymerase be the archaeal dna polymerase of shortage 5 '-3 ' exonuclease activity of the hard genus bacillus of self-heating, and described ribonuclease H is coli rnase enzyme H.

Be prepared as follows reaction mixture:

One or two primer of 35mM tris-hydrochloride buffer reagent (pH 7.5), 0.1mg/ml bovine serum albumin (BSA), 2.7% glycerine, 5% methyl-sulphoxide, the various dNTP of 1.4mM, 10mM magnesium chloride, 20pM above-mentioned (1) described primer centering, 0.6ng are used as synthesizing single-stranded DNA, 5U Bca BEST archaeal dna polymerase and the 60U clone's of template ribonuclease H, and the end reaction volume is 50 μ l.Make the reaction mixture homogeneous, in 55 ℃ of incubations 60 minutes, then in 2 minutes described enzymes of deactivation of 90 ℃ of heating.Get 8 μ l reaction mixtures and on 3: 1 agaroses (Takara Shuzo) of 3%NuSieve gel, carry out electrophoresis.Below be presented in the primer that uses in the corresponding model:

Model 1-5: use primer a pair of among the 1-5;

Model 6: only use primer to 2 downstream primer; And

Model 7: use primer not add ribonuclease H to 4.

The result observes and has the amplified fragments of 40 base pairs (bp) to the purpose magnitude range of about 90bp when the reaction mixture of the 2-5 that uses a model, illustrates that DNA utilizes these reactive systems to increase.In only using two kinds of primers, observe the amplified fragments (single stranded DNA fragment) of expection size in a kind of model 6 of primer with about 70 bases (b).In the reaction of model 1 or 7, do not observe DNA cloning.

(3) confirm amplified production

Filter the reaction mixture that above-mentioned (2) described model 4 reactions obtain with Microcon-100 (Takara Shuzo), reclaim the amplification of DNA fragments that is captured on the filter membrane.Utilize dideoxy method to record the nucleotide sequence of dna fragmentation.The result confirms that the fragment of above-mentioned reaction amplification is the DNA with nucleotide sequence identical with template DNA.

(4) the detection reaction time

The reaction mixture for preparing above-mentioned (2) described model 2 is with the variation of research amplified production amount when reacting different time.Make reaction mixture in 55 ℃ of incubations 0,15,30,60,90 or 120 minutes.Then in 90 ℃ for the treatment of mixture 2 minutes, the described enzyme of deactivation.Get 8 μ l reaction mixtures and sepharose on carry out electrophoretic analysis at 3: 1 at 3%NuSieve.Electrophoresis result is seen Fig. 2.Digital 1-6 represents 0,15,30,60,90 or 120 minute reaction mixture swimming lane of point sample reaction respectively among the figure.M represents the swimming lane of point sample as the 100bpDNA ladder type mark (Takara Shuzo) of molecular weight marker.

As shown in Figure 2,0 minute reaction times did not observe amplified production.Confirmation is along with the time lengthening of reaction times from 15 minutes to 30 or 60 minutes, and the amplified production amount increases.Yet it is 60 minutes or almost not variation of amplified production amount for more time that electrophoresis observes the reaction times, illustrates that the amplification of used reactive system reached platform in the time of about 60 minutes.

Embodiment 2

(1) preparation RNA

Use TRIzol reagent (Life Technologies), be used as the RNA of template with people's cell HT29 (ATCC HTB-38) (Dainippon Pharmaceutical) preparation of cultivating in the present embodiment.The concentration adjustment of the total RNA that obtains is 1 μ g/ μ l.The OD260/OD280 value is 1.8, and it represents the spectrophotometric purity of RNA.

(2) amplified reaction

Bca BEST archaeal dna polymerase and ribonuclease H endonuclease that use has reverse transcriptase activity and dna polymerase activity determine whether by RNA amplification cDNA.

Add the above-mentioned total RNA of 1 μ g and prepare reaction mixture with embodiment 2 described compositions.The primer that utilizes embodiment 1 is to 2 target areas (gene accession number X01060) as primer amplification coding human transferrin acceptor.

Make reaction mixture in 55 ℃ of incubations 60 minutes, then in 2 minutes described enzymes of deactivation of 90 ℃ of heating.When 8 μ l reaction mixtures during electrophoresis, observe the amplified fragments with 56bp expection size on 3: 1 sepharoses of 3%NuSieve.In addition, carry out RNA hybridization with probe with target nucleotide sequence.Use 5 '-terminal dna probe with the nucleotide sequence shown in the biotin labeled SEQ ID NO:4 with sequence table carries out DNA hybridization.The hybridization of the fragment of described probe of result and above-mentioned amplification confirms the inventive method target area that correctly increases.

Embodiment 3

(1) synthetic primer

Use double-stranded DNA as template study amplification method of the present invention.With the synthetic used primer of dna synthesizer (Applied Biosystems).The basic nucleotides sequence of primer is shown in the SEQ ID NO:5-13 of sequence table.Below be presented in the structure of the primer of present embodiment use in detail.Use pUC19 DNA (Takara Shuzo) as the template of primer to A-F.Can obtain the nucleotide sequence (gene accession number L09137) of pUC19 from database.The double chain DNA fragment that is increased is as the template of primer to G.The primer and TaKaRa RNA PCR test kit (AMV) Ver.2.1 (Takara Shuzo) that use the sequence shown in the SEQ ID NO:14 or 15 with sequence table according to appended standard method prepare fragment by the human total rna that embodiment 2 obtains.

Primer is to A (expanding fragment length: about 450bp): have wherein 3 '-combination of primers of the nucleotide sequence shown in the SEQ ID NO:5 or 6 of the sequence table that terminal first and second base are replaced by ribonucleotide;

Primer is to B (expanding fragment length: about 250bp) have wherein 3 '-combination of primers of the nucleotide sequence shown in the SEQ ID NO:5 or 7 of the sequence table that terminal first and second base are replaced by ribonucleotide;

Primer is to C (expanding fragment length: about 520bp): have wherein 3 '-combination of primers of the nucleotide sequence shown in the SEQ ID NO:5 or 8 of the sequence table that terminal first and second base are replaced by ribonucleotide;

Primer is to D (expanding fragment length: about 890bp): have wherein 3 '-combination of primers of the nucleotide sequence shown in the SEQ ID NO:5 or 9 of the sequence table that terminal first and second base are replaced by ribonucleotide;

Primer is to E (expanding fragment length: about 130bp): have wherein 3 '-combination of primers of the nucleotide sequence shown in the SEQ ID NO:10 or 6 of the sequence table that terminal first to the 3rd base replaced by ribonucleotide;

Primer is to F (expanding fragment length: about 220bp): have wherein 3 '-combination of primers of the nucleotide sequence shown in the SEQ ID NO:11 or 6 of the sequence table that terminal first to the 3rd base replaced by ribonucleotide; And

Primer is to G (expanding fragment length: about 320bp): have wherein 3 '-combination of primers of the nucleotide sequence shown in the SEQ ID NO:12 or 13 of the sequence table that terminal first to the 3rd base replaced by ribonucleotide.

(2) amplified reaction

Be prepared as follows reaction mixture:

Each primer of 35mM potassium phosphate buffer agent (pH 7.5), 0.1mg/ml bovine serum albumin (BSA), 5% methyl-sulphoxide, the various dNTP of 1.4mM, 10mM magnesium chloride, 60pM above-mentioned (1) described primer centering, 100ng are used as pUC19 DNA, 5.5U Bca BEST archaeal dna polymerase and the 60U ribonuclease H of template, and the end reaction volume is 50 μ l.

Reaction conditions is as follows.Make the reaction mixture that do not have archaeal dna polymerase or ribonuclease H in 98 ℃ of heat denatured 1 minute, be cooled to 55 ℃ then.Wherein add archaeal dna polymerase and ribonuclease H then, mixture was in 55 ℃ of incubations 60 minutes.After finishing reaction, make mixture in 2 minutes described enzymes of deactivation of 90 ℃ of heating.Get 8 μ l reaction mixtures then and sepharose on carry out electrophoresis at 3: 1 at 3%NuSieve.

The result confirms to use any one primer to all obtaining the purpose amplified fragments.Therefore confirm that double-stranded DNA can carry out amplified reaction in the amplification method of the present invention as template.

(3) with restriction enzyme digest amplification product

The detection limit enzyme is to the digestion of the amplified fragments that obtains with amplification method of the present invention.The pUC19 plasmid DNA is as template DNA.Use the SEQ ID NO:5 of sequence table and pUC19 upstream (2) NN primer and the pUC19 downstream NN primer shown in 6 difference.In described various primers, first of 3 '-end and second base are replaced by ribonucleotide.Reaction mixture composed as follows.

Reaction mixture A:35mM potassium phosphate buffer agent (pH 7.5), 10mM magnesium chloride, the various dNTP of 1.4mM, 0.01%BSA, 5%DMSO, 2.7% glycerine, each pUC19 upstream (2) NN primer of 100pM and pUC19 downstream NN primer, 500ng pUC19DNA, adding sterile distilled water to reaction volume is 48 μ l.

Reaction mixture is cooled to 55 ℃ then in 98 ℃ of heat denatured 1 minute.To make reaction volume be 50 μ l to wherein adding 60U coli rnase enzyme H and 5.5U Bca BEST then.Make reaction mixture in 55 ℃ of incubations 1 hour.After finishing reaction, make mixture in 2 minutes described enzymes of deactivation of 90 ℃ of heating.Make reaction mixture on 3% sepharose, carry out electrophoresis then, the amplified production that purifying obtains.The amplified production that is reclaimed is suspended in the 100 μ l sterile distilled waters again.

Use the dna solution that so obtains to carry out restriction enzyme digestion.Used restriction enzyme is AccII (Takara Shuzo) and BcnI (Takara Shuzo).Reaction mixture composed as follows.

3 μ l dna solutions, the incidental 10 * AccII damping fluid of 1 each enzyme of μ l or 10 * BcnI damping fluid, 1 μ l restriction enzyme AccII or BcnII, adding sterile distilled water to reaction volume is 10 μ l.Reaction mixture was in 37 ℃ of reactions 30 minutes.To wherein adding 1.5 μ l, 10 * sample loading buffer.Get 6 μ l mixtures and on the 3%NuSieve sepharose, carry out electrophoresis.

The result all obtains the target DNA fragment of restriction enzyme digestion with two kinds of restriction enzyme AccII and BcnI.

(4) detect sudden change

Research and utilization amplification method of the present invention detects sudden change.PUC19 is as template.The basic nucleotide sequence of pUC19 upstream (2) NN primer and pUC19 downstream NN primer is shown in the SEQ ID NO:5 and 6 of sequence table respectively.These two kinds of primers are wherein 3 '-first terminal and second chimeric oligonucleotide primer that base is replaced by Yeast Nucleic Acid.In addition, prepare 4 kinds of such primers: the base of pUC19 upstream (2) NN primer 3 '-end is replaced by U (the corresponding base complementrity in it and the template) or A, C or G (it is a base mismatch), is called pUC19 upstream (2) NN-U, pUC19 upstream (2) NN-A, pUC19 upstream (2) NN-C or pUC19 upstream (2) NN-G.The combination of these primers is as follows:

Primer is to 1:pUC19 upstream (2) NN-U and pUC19 downstream NN;

Primer is to 2:pUC19 upstream (2) NN-A and pUC19 downstream NN;

Primer is to 3:pUC19 upstream (2) NN-C and pUC downstream NN; And

Primer is to 4:pUC19 upstream (2) NN-G and pUC19 downstream NN.

Be prepared as follows reaction mixture.

30mM potassium phosphate buffer agent (pH 7.3), 0.01% bovine serum albumin (BSA), the various dNTP of 5%DMSO, 1mM, 8mM magnesium acetate, each primer of 60pM, 50ng template DNA, adding sterile distilled water to reaction volume is 48 μ l.

Reaction mixture is cooled to 55 ℃ then in 98 ℃ of heat denatured 1 minute.Then to wherein adding 5.5U Bca BEST archaeal dna polymerase and 60U coli rnase enzyme H, make reaction mixture in 55 ℃ of incubations 60 minutes.Make mixture in 2 minutes described enzymes of deactivation of 90 ℃ of heating then.Get 8 μ l reaction mixtures and on 3: 1 agaroses (TakaraShuzo) of 4%NuSieve gel, carry out electrophoresis.The result confirms to have only when using the 3 '-end that is included in pUC19 upstream (2) NN to have the combination of primers of the primer of complementary base, just detects the purpose amplified fragments of about 450bp.On the contrary, in having the combination of primer of base mismatch, the 3 '-end that is included in pUC19 upstream (2) NN do not observe amplified fragments.

Embodiment 4

(1) reaction of usefulness microtest tube

The reaction volume of research amplification method of the present invention.The section of selecting coding human transferrin acceptor is as the amplification section.Use has the primer of sequence shown in the SEQ ID NO:12 or 13 of sequence table.In described primer, first of 3 '-end and second base are replaced by ribonucleotide.The fragment of about 750bp of RT-PCR amplification is as template DNA.Reaction volume is adjusted to 50,100,300 or 500 μ l.Reaction mixture is composed as follows.

The Bca BEST archaeal dna polymerase (22U/ μ l) of 10 μ M ATP, the 1 μ l of the 100mM magnesium acetate of the 5 * special damping fluid of reaction mixture A:10 μ l (135mM potassium phosphate buffer agent (pH 7.5), 0.5mg/ml BSA, 2.5%DMSO), 4 μ l, the various dNTP of 10mM, the 10 μ l of 5 μ l, 1 μ l ribonuclease H (60U/ μ l) add sterile distilled water to 39 μ l.

Reaction mixture B: the dna profiling of the 20 μ M human transferrin acceptor S primers (SEQ IDNO:12) of each 3 μ l and 20 μ M human transferrin acceptor primers (SEQ ID NO:13), about 100ng adds sterile distilled water to 11 μ l.If reaction volume is 50 μ l or higher, increase said components in proportion.

For amplified reaction, reaction mixture B was handled 2 minutes in 98 ℃, then in 55 ℃ of incubations 3 minutes.Reaction mixture B is added in the 1500-μ l microtest tube in the reaction mixture A of 55 ℃ of preincubation.After the mixing, make reaction mixture in 55 ℃ of incubations 1 hour.After finishing reaction, mixture is transferred in the ice bath.Get 8 μ l reaction mixtures and on 3% sepharose, carry out electrophoresis.

The result adopts effectively the increase purpose fragment of about 300bp of each described volume.In addition, confirm to adopt the fragment of pcr amplification to be used as the purpose fragment that template DNA can obtain amplification without any problem ground.

(2) reaction of usefulness Petri dish

Research uses Petri dish to prevent because the heterogeneity of the reaction mixture temperature due to the reaction volume increase.The section of selecting coding human transferrin acceptor is as the amplification section.Use has the primer of sequence shown in the SEQ ID NO:12 or 13 of sequence table.In described primer, first of 3 '-end and second base are replaced by ribonucleotide.The fragment of about 750bp of RT-PCR amplification is as template DNA.Reaction volume is adjusted to 10ml.Reaction mixture is composed as follows.

5 * special the damping fluid of reaction mixture A:2000 μ l (135mM potassium phosphate buffer agent (pH 7.5), 0.5mg/ml BSA, 2.5%DMSO), the 100mM magnesium acetate of 800 μ l, the various dNTP of 10mM of 1000 μ l add sterile distilled water to 9.1ml.

Reaction mixture B: the DNA as template of the 60 μ M human transferrin acceptor S primers (SEQ IDNO:12) of each 200 μ l and 60 μ M human transferrin acceptor primers (SEQ ID NO:13), about 10 μ g adds sterile distilled water to 500 μ l.

Reaction mixture C:200 μ l Bca BEST archaeal dna polymerase (22U/ μ l) and 200 μ l ribonuclease Hs (60U/ μ l).

For amplified reaction, reaction mixture B was handled 1 minute in 98 ℃, then in 55 ℃ of incubations 3 minutes.Reaction mixture B is added in 60-mm (diameter) the plastics Petri dish in the reaction mixture A of 55 ℃ of preincubation.Reaction mixture C adds wherein again.After the mixing, make reaction mixture in 55 ℃ of incubations 1 hour.After finishing reaction, mixture is transferred in the ice bath.Get 8 μ l reaction mixtures and on 3% sepharose, carry out electrophoresis.

The purpose fragment of about 300bp even result's employing 10ml reaction volume also effectively increases.In addition, confirm to adopt the fragment of pcr amplification to be used as the purpose fragment that template DNA can obtain amplification without any problem ground.Therefore confirm to compare with conventional PCR method, method of the present invention can more preferably be used to prepare the DNA chip of a large amount of dna fragmentations of needs.

Embodiment 5

(1) relation between used buffer type and the ribonuclease H amount

The relation of institute between buffer type and the ribonuclease H amount.Wherein the fragment cloning of 249bp or 911bp is gone into the plasmid DNA of pUC19 carrier (being called pUC19-249 and pUC19-911) as template.Chimeric oligonucleotide primer is as primer, and first is replaced by ribonucleotide to the 3rd base to have 3 ' of MF2N3 (24) primer of the SEQ ID NO:16 of sequence table or 17 described sequences or MR1N3 (24) primer-terminal in described primer.Use these combination of primers to obtain the about 450bp of pUC19-249 and pUC19-911 and the amplified fragments of about 1100bp respectively.

Select tris-hydrochloride damping fluid, potassium phosphate buffer and Tricine damping fluid as buffering system to be studied.Wherein do not add the ribonuclease H amount of being studied, its final concentration scope is 0.3-1.2U/ μ l.As described in embodiment 1 (2), prepare tris-hydrochloride buffering system, the different Bca BEST archaeal dna polymerases that is to use 10ng pUC19-249 or each described primer of 200ng pUC19-911,60pM and 11U/50 μ l reaction volume.Preparation contains the potassium phosphate buffer system of similar composition.Preparation contains the Tricine Laemmli buffer system Laemmli of each composition of final concentration shown below: 34mM Tricine buffer reagent (pH 8.7), 10mM sodium-chlor, 10mM ammonium sulfate, 0.01%BSA, 1%DMSO, 4mM magnesium acetate and each dNTP of 0.5mM.The pUC19-249 plasmid or the pUC19-911 plasmid of 200ng/50 μ l reaction volume, each primer of 60pM/50 μ l reaction volume, the RNA enzyme H of predetermined concentration and the Bca BEST archaeal dna polymerase of 11U/50 μ l reaction volume that in described buffering system, add 10ng/50 μ l reaction volume.

For amplified reaction, in 98 ℃ of heat denatured 1 minute, be cooled to 55 ℃ then as the mixture of the pUC19-249 of template or pUC19-911 and corresponding primer.To wherein adding the remaining reaction mixture of ingredients.Make mixture in 55 ℃ of reactions 60 minutes.After reaction is finished, make mixture be cooled to 4 ℃, to the 0.5M EDTA stopped reaction that wherein adds 1/10 volume.Get 3 μ l reaction mixtures and on 3: 1 agaroses (Takara Shuzo) of 3%NuSieve gel, carry out electrophoresis.

The result when pUC19-249 when the template, it is as follows that used buffering system is observed amplification efficiency increase order: tris-hydrochloride＜potassiumphosphate＜Tricine.When pUC19-911 was used as template, the amplification efficiency increase order that used buffering system is observed was as follows: tris-hydrochloride＜Tricine＜potassiumphosphate.The result does not add ribonuclease H and does not observe the purpose amplified fragments, is the ribonuclease H generation purpose amplified fragments of 0.3-1.2U/ μ l but be to use final concentration.

(2) research primer amount

Research the primer amount is to the influence of amplification method of the present invention.Use has wherein in above-mentioned (1) described component pUC19-249 as the reaction mixture system of the composition of template.The ribonuclease H of 60U/50 μ l reaction volume is used for the potassiumphosphate buffering system, and the ribonuclease H of 30U/50 μ l reaction volume is used for tris-hydrochloride buffering system or Tricine buffering system.The primer concentration scope of being studied is 10-100pM/50 μ l.The confirmation of reaction conditions and amplification is as described in above-mentioned (1).

The result with contain the primer concentration scope be 10-100pM/50 μ l respectively react the purpose fragment that buffering system observes amplification.

(3) influence of the pH of reaction buffer

The pH of research reaction mixture is to the influence of amplification method of the present invention.The composition of reaction mixture is as described in above-mentioned (2).The pH of the potassiumphosphate buffering system of being studied is 7.0-8.0, and the pH of Tricine buffering system is 7.5-9.2, and the pH of tris-hydrochloride buffering system is 7.5-9.0.The confirmation of reaction conditions and amplification is as described in above-mentioned (1).

The result observes the purpose fragment of amplification in the pH scope that is used for corresponding buffering system.

(4) influence of additive

The reaction mixture composition research of using above-mentioned (3) described phosphoric acid buffer systems (pH 7.5) adds the influence of methyl-sulphoxide (DMSO).In addition, the also effect of research adding polyamine.The amount from 0 to 10% of the adding DMSO that is studied.On the other hand, four hydrochloric acid spermine (Sigma), Spermidine hydrochloride (Sigma), acetyl putrescine (Nacalai Tesque), disalt sour amine (NacalaiTesque), trimethylene diamine (Nacalai Tesque), propylene diamine (Nacalai Tesque) and two hydrochloric acid diamino methane (Nacalai Tesque) are as polyamine.The propylene diamine and the trimethylene diamine weight range that add are 0-2%.The amount ranges of other polyamine is 0-5mM.The confirmation of reaction conditions and amplification is as described in above-mentioned (1).

The additive that the result uses scope concentration shown below target DNA fragment: the 0-5%DMSO that effectively increases; 0-200 μ M four hydrochloric acid spermine or spermidines; 40 μ M-40mM acetyl putrescine or disalt sour amine; The 0.002%-0.02% trimethylene diamine; 0.0001%-0.01% propylene diamine and 0.1 μ M-10 μ M, two hydrochloric acid diamino methane.

(5) research magnesium salts type

Research magnesium salts type is to the influence of amplification method of the present invention.PUC19 DNA is as template.Have pUC19 upstream NN 249 primers of sequence shown in the SEQ ID NO:11 and 6 of sequence table and pUC19 downstream NN primer respectively as primer.Use the amplified fragments that a pair of such primer obtains about 225bp.Magnesium chloride, magnesium acetate and sal epsom are as magnesium salts.Reaction mixture composed as follows.

The Bca BEST archaeal dna polymerase of 35mM potassium phosphate buffer agent (pH 7.3), 8mM (final concentration) magnesium chloride, magnesium acetate or sal epsom, each dNTP of 1.0mM (final concentration), 50ng pUC19 DNA, each primer of 60pM, 60U ribonuclease H, 5.5U, adding sterile distilled water to reaction volume is 50 μ l.The confirmation of reaction conditions and amplification is as described in above-mentioned (3).

The result observes the purpose amplified fragments with each described magnesium salts.

(6) concentration of research magnesium and dNTP

Research magnesium and dNTP concentration are to the influence of amplification method of the present invention.The composition of reaction mixture is as described in above-mentioned (5), the different pUC19 DNA that is to use 25ng and the magnesium and the various dNTP of different concns.The confirmation of reaction conditions and amplification is as described in above-mentioned (1).

Be fixed as in the reactive system of 1mM at each dNTP final concentration,, obtain the purpose amplified fragments when the magnesium final concentration scope of using during as 6mM-10mM.Be fixed as in the reactive system of 8mM at the magnesium final concentration,, obtain the purpose amplified fragments when each dNTP final concentration scope of using during as 0.6mM-1.2mM.In addition, be fixed as in the reactive system of 0.5mM,, obtain the purpose amplified fragments when the magnesium final concentration scope of using during as 2mM-6mM in each dNTP final concentration scope.Be fixed as in the reactive system of 4mM at the magnesium final concentration,, obtain the purpose amplified fragments when each dNTP final concentration scope of using during as 0.2mM-0.8mM.

(7) research potassium phosphate buffer agent or Tricine buffer concentration change and are reactive

Research potassium phosphate buffer agent or Tricine buffer concentration are to the influence of amplification method of the present invention.As under the situation of template, the composition of reaction mixture is described with above-mentioned (1) at pUC19-249, and the different final concentrations that are to use are the potassium phosphate buffer agent of 20-50mM or the Tricine buffer reagent that final concentration is 22-46mM.The confirmation of reaction conditions and amplification is as described in above-mentioned (1).

The result is when using the final concentration scope during as the Tricine buffer reagent of 22-46mM, to obtain the purpose amplified fragments as the potassium phosphate buffer agent of 20-50mM or final concentration scope.

(8) concentration of research Bca BEST archaeal dna polymerase

Research Bca BEST archaeal dna polymerase concentration is to the influence of amplification method of the present invention.As under the situation of template, the composition of reaction mixture is described with above-mentioned (1) at pUC19-249, and different be to use potassiumphosphate buffering systems or Tricine buffering system and concentration range are the Bca BEST archaeal dna polymerase of 1-22U/50 μ l reaction volume.The confirmation of reaction conditions and amplification is as described in above-mentioned (1).

When the result is the Bca BEST archaeal dna polymerase of 1-22U/50 μ l when the working concentration scope, obtain the purpose amplified fragments.

Embodiment 6

Comparison with PCR method

Compare amplification method of the present invention and PCR method.The dna fragmentation of about 150bp or about 250bp inserts the DNA of pUC19 plasmid DNA multiple clone site as template.Be prepared as follows template.

PUC19 upstream 150 primers, pUC19 upstream 249 primers and the pUC19 downstream NN primer that has sequence shown in the sequence table SEQ ID NO:10,11 and 6 respectively is used for utilizing the pUC19 plasmid DNA of 100pg to carry out the PCR reaction as template.Adopt the amplified fragments of the about 150bp of combination acquisition of pUC19 upstream 150 primers and pUC19 downstream NN primer.Adopt the amplified fragments of the about 250bp of combination acquisition of pUC19 upstream 249 primers and pUC19 downstream NN primer.Use each described amplified fragments of Microcon-100 purifying of DNA flush end test kit (Takara Shuzo) flush end end, make it the HincII site that subclone is gone into the pUC19 plasmid then.The plasmid that wherein inserts a kind of described amplified fragments is used for transformed into escherichia coli JM109.Cultivate the transformant that obtains, use qiagen plasmid trace quantity reagent kit (Qiagen) and have the plasmid that inserts DNA from described Bacillus coli cells purifying.Have the plasmid that inserts DNA and be used as template.

The primer sequence of Shi Yonging is shown in the SEQ ID NO:18 and 19 of sequence table in the present embodiment.Wherein first of 3 '-end to the 3rd primer that base is replaced by ribonucleotide is used for amplification method of the present invention.Reaction mixture composed as follows.

27mM phosphate buffer (pH 7.3), 0.01% bovine serum albumin (BSA), each dNTP of 5%DMSO, 1mM, 8mM magnesium acetate, each primer of 60pM, 1ng template DNA, adding sterile distilled water to reaction volume is 48 μ 1.

Reaction mixture is cooled to 55 ℃ then in 98 ℃ of heat denatured 1 minute.Then to wherein adding 5.5U Bca BEST archaeal dna polymerase and 60U coli rnase enzyme H, make mixture in 55 ℃ of incubations 60 minutes.Make mixture in 2 minutes described enzymes of deactivation of 90 ℃ of heating then.Get 3 μ l reaction mixtures and on 3: 1 agaroses (Takara Shuzo) of 4%NuSieve gel, carry out electrophoresis.

On the other hand, use the amplification of PCR method in contrast.Described reactive applications pcr amplification test kit (Takara Shuzo), 10pM have each primer that does not contain ribonucleotide, the 1ng template DNA of sequence shown in the SEQ ID NO:18 or 19 of sequence table, and adding sterile distilled water to reaction volume is 50 μ l.Reaction conditions is 94 ℃ 30 seconds, 55 ℃ of 25 round-robin 30 seconds and 72 ℃ 40 seconds.After finishing reaction, get 3 μ l reaction mixtures at 4%NuSieve3: carry out electrophoresis on 1 agarose (Takara Shuzo) gel.

The result compares with PCR method, is inserted segmental each plasmid amplification and is obtained more purpose fragment by have 150bp or 249bp as template in amplification method of the present invention.With Microcon-100 purifying 20 μ l reaction mixtures, use the amount of Beckman DU-640 spectrophotometer (Beckman) quantitative amplification product, so that with the amount of numeral amplified production.Confirmation in amplification method of the present invention by as template to have the fragment amount that 150bp inserts segmental plasmid amplification higher about 60 times than PCR method.Also confirm in amplification method of the present invention by as template to have the fragment amount that 250bp inserts segmental plasmid amplification higher about 40 times than PCR method.Confirm that based on these results compare with conventional PCR method, method of the present invention can more preferably be used to prepare the DNA chip of a large amount of dna fragmentations of needs.

Embodiment 7

(1) preparation rna probe

Research is used to detect the method for the amplified fragments that obtains with amplification method of the present invention.Preparation wherein two kinds of different fluorescent substances is connected to the detection probes that the ribonucleotide of two ends of probe is formed.Use the synthetic rna probe that is used to detect of dna synthesizer (Applied Biosystems).The nucleotides sequence of described probe is shown in the SEQ ID NO:20 of sequence table.6-FAM (Glen Research) and TAMRA (Glen Research) are used separately as in the fluorescent substance of 5 '-end and 3 '-end mark probe.

(2) amplified reaction and detection

0.1 or the pUC19 DNA of 1ng is as template.Wherein 3 ' of primer terminal first and second base are replaced, have respectively pUC19 upstream 150 primers of sequence shown in the SEQ ID NO:10 and 8 of sequence table and pUC19 downstream 542 primers as primer by ribonucleotide.

Reaction mixture composed as follows.

27mM phosphate buffer (pH 7.3), 0.01%BSA, 5%DMSO, each dNTP of 1mM, 8mM magnesium acetate, each described primer of 60pM, 0.1 or the template DNA of 1ng, the rna probe of 0.1 μ g add sterile distilled water to 48 μ l reaction volume.Also prepare with the reaction mixture that does not have template DNA that compares.

Make reaction mixture in 98 ℃ of heat denatured 1 minute, be cooled to 55 ℃ then.To wherein adding 22U Bca BEST archaeal dna polymerase or sterilized water and 60U coli rnase enzyme H, make mixture in 55 ℃ of incubations 60 minutes.In described mixture, add 5 μ l10% sodium lauryl sulphate (SDS then; Nacalai Tesque) the described enzyme of deactivation.Dilute 50 μ l reaction mixtures with isopyknic sterilized water, then it is transferred in the micro plate.Application image analyser FM BIO II Multi-View (Takara Shuzo) detects in excitation wavelength 505nm.

The result adopts wherein any template all not detect fluorescent signal when not adding Bca BEST archaeal dna polymerase.When not adding template DNA, in the reaction mixture that contains Bca BEST archaeal dna polymerase, do not detect fluorescent signal yet.On the other hand, when adding the template DNA of 0.1ng or 1ng, all detect fluorescent signal.In addition, have only when under having Bca BEST archaeal dna polymerase, adding 0.1ng or 1ng template DNA, just containing the purpose amplified fragments that observes about 190bp on 3% sepharose of 0.00003% ethidium bromide through electrophoresis.That is to say that the detection method of using rna probe obtains identical result with conventional electrophoretic detection.Therefore, set up and be used to detect the method for rna probe of using by the amplified fragments of amplification method acquisition of the present invention.

Embodiment 8

The primer that the research deoxyribonucleotide is formed is as one of described two kinds of primers in the inventive method.The M4 primer (Takara Shuzo) that has the MR1N3 (30) of sequence shown in the SEQ ID NO:19 of sequence table and have a sequence shown in the SEQ ID NO:58 of sequence table is as primer.In the MR1N3 primer, first of 3 '-end to the 3rd base replaced by ribonucleotide.Reaction mixture composed as follows.

The template DNA of 27mM phosphate buffer (pH 7.3), 0.01% bovine serum albumin (BSA), each dNTP of 5%DMSO, 1mM, 8mM magnesium acetate, each primer of 30pM, 1ng adds sterile distilled water to 24 μ l reaction volume.

Reaction mixture is cooled to 55 ℃ then in 98 ℃ of heat denatured 2 minutes.Make that to wherein adding 11U Bca BEST archaeal dna polymerase and 30U coli rnase enzyme H reaction volume is 25 μ l.Make reaction mixture in 55 ℃ of incubations 60 minutes.Make mixture in 2 minutes described enzymes of deactivation of 90 ℃ of heating then.Get 5 μ l reaction mixtures and sepharose on carry out electrophoresis at 3: 1 at 4%NuSieve.Result viewing is to the purpose amplified fragments.

Embodiment 9

Method of the present invention is used for detecting blood group intestinal bacteria O-157.

The sequence of the primer of Shi Yonging is shown in the SEQ ID NO:21-24 of sequence table in the present embodiment.In order to detect Vero cytotoxin 1 or Vero cytotoxin 2 sequences of coding intestinal bacteria O-157, according to the introduction (Clinical Microbiology) of Rinsho To Biseibutsu, 18 (4): 507-513 (1991) makes up the combination of primers of the sequence with SEQ ID NO:21 or 22 and has the combination of primers of the sequence of SEQ ID NO:23 or 24.Wherein first of 3 '-end to the 3rd primer that base is replaced by ribonucleotide is used for amplification method of the present invention.The following heat extraction substrate that makes is used as template: the culture of results bleeding type intestinal bacteria O-157 (ATCC preserving number 43895), suspend with suitable cell density with sterilized water, and handled 10 minutes in 98 ℃.Reaction mixture composed as follows.

27mM phosphate buffer (pH 7.3), 0.01% bovine serum albumin (BSA), each dNTP of 5%DMSO, 1mM, 8mM magnesium acetate, each primer of 60pM, be equivalent to 10 ⁴-10 ⁶The template DNA of cell (heat extraction substrate) adds sterile distilled water to 48 μ l reaction volume.

Reaction mixture is cooled to 55 ℃ then in 98 ℃ of heat denatured 1 minute.To wherein adding 5.5U Bca BEST archaeal dna polymerase and 60U coli rnase enzyme H.Make reaction mixture in 55 ℃ of incubations 60 minutes.Make mixture in 2 minutes described enzymes of deactivation of 90 ℃ of heating then.Get 3 μ l reaction mixtures and on 3: 1 agaroses (Takara Shuzo) of 4%NuSieve gel, carry out electrophoresis.

The result uses any a pair of of described two kinds of primer centerings and is equivalent to 10 ⁴The template DNA of cell can detect O-157 Vero cytotoxin 1 and 2, confirms that method of the present invention can be used as the method that detects poisonous bacterium.

Embodiment 10

Study the amplification of method of the present invention to the long-chain dna fragmentation.Be prepared as follows double-stranded DNA as template.At first, use Uni-ZAP XR carrier (Stratagene) according to ordinary method and make up the library by the mRNA that obtains from normal gastric mucosa.The screening library is to select to have the segmental clone of insertion of about 2.1kbp or about 4.3kbp.The clone is used for obtaining pBluescript SK (-) by external cutting and has a liking for the thalline carrier.Use plasmid, have an amplified fragments that the MCR-F primer of sequence shown in sequence table SEQ ID NO:25 and 26 and MCR-R primer and pcr amplification test kit (Takara Shuzo) obtain about 2.2kbp and about 4.4kbp respectively as template.These PCR fragments are as the template of amplification method of the present invention.Wherein first of 3 '-end to the 3rd base is used as primer by MF2N3 (24) primer and MR1N3 (24) primer that ribonucleotide replaces, has respectively sequence shown in sequence table SEQ ID NO:27 and 28.Reaction mixture composed as follows.

28mM phosphate buffer (pH 7.5), 0.01% bovine serum albumin (BSA), each dNTP of 1%DMSO, 0.5mM, 4mM magnesium acetate, each primer of 30pM, 0.2mM putrescine add sterile distilled water to 24.25 μ l.Reaction mixture is cooled to 55 ℃ then in 92 ℃ of processing 2 minutes.Make reaction volume to 25 μ l to wherein adding 30U ribonuclease H and 5.5U Bca BESTDNA polysaccharase.Made the reaction mixture incubation 1 hour.After finishing reaction, mixture is in 4 ℃ of coolings, to the 0.5M EDTA solution stopped reaction that wherein adds 2.5 μ l.Get 5 μ L mixtures and on 1% sepharose, carry out electrophoresis.

The result obtains the amplified fragments of about 2.2kbp or about 4.4kbp with method of the present invention, confirms that method of the present invention can be used for the long-chain dna fragmentation that increases.

Embodiment 11

Produce the dna microarray of λ dna fragmentation point sample on it of the 300bp of the λ dna fragmentation of about 400bp of amplification method of the present invention amplification and pcr amplification and 1000bp.Obtain the nucleotide sequence of λ DNA by GenBank accession number V00636, J02459, M17233 and X00906.The sequence of the primer of Shi Yonging is shown in sequence table SEQ IDNO:25-26 and 29-35 in the present embodiment.Be prepared as follows the reaction mixture that is used for amplification method of the present invention.

34mM Tricine-hydrochloride buffer reagent (pH 8.7), 10mM Repone K, 10mM ammonium sulfate, 0.01% bovine serum albumin (BSA), 1% methyl-sulphoxide, 4mM magnesium acetate, each dNTP of 0.5mM, each primer of 500pM, 100ng are used as pcr amplification product, 110U Bca BEST archaeal dna polymerase and the 300U clone's of template ribonuclease H, and the end reaction volume is 500 μ l.Reaction mixture is to homogeneous, in 55 ℃ of incubations 60 minutes, then in 2 minutes described enzymes of deactivation of 90 ℃ of heating.This solution is used for step subsequently.The dna fragmentation of point sample is as follows.

1. sample: will use λ DNA and make template and have pcr amplification product (300bp) subclone that the combination of primers of sequence shown in sequence table SEQ ID NO:29 or 30 obtains to go in the pUC19 carrier.Utilize then and have the primer of sequence shown in sequence table SEQ ID NO:25 or 26 through the described subclone product of pcr amplification.Thus obtained product as first and second base of template and wherein said primer 3 '-end by ribonucleotide replacement, chimeric oligonucleotide primer with sequence shown in sequence table SEQ ID NO:31 or 32, be used for product by the about 400bp of amplification method of the present invention amplification, with the acquisition sample.5 kinds of dna solutions are used for point sample, and described dna solution is an original content reaction mixture or with the reaction mixture of 2 times, 4 times, 8 times of carbonate buffer solution (using the carbonate buffer solution of 50mM concentration to dilute under each situation) dilutions or 16 times.

2. sample: usefulness Microcon-100 (Takara Shuzo) handles above-mentioned 1 dna fragmentation that increases.Be that 0.125 μ g/ μ l, 0.25 μ g/ μ l, 0.5 μ g/ μ l, 1.0 μ g/ μ l and 2.0 μ g/ μ l make 5 kinds of dna solutions with the 50mM carbonate buffer solution with concentration adjustment then.

3. positive control: handle with Microcon-100 and to use λ and have a liking for thallus DNA as template with have the pcr amplification product (300bp) that the combination of the primer of sequence shown in sequence table SEQ ID NO:29 or 30 obtains.Be that 0.125 μ g/ μ l, 0.25 μ g/ μ l, 0.5 μ g/ μ l, 1.0 μ g/ μ l and 2.0 μ g/ μ l make 5 kinds of dna solutions with the 50mM carbonate buffer solution with concentration adjustment then.

4. positive control: handle with Microcon-100 and to use λ and have a liking for thallus DNA as template with have the pcr amplification product (1000bp) that the combination of the primer of sequence shown in sequence table SEQ ID NO:33 or 34 obtains.Be that 0.125 μ g/ μ l, 0.25 μ g/ μ l, 0.5 μ g/ μ l and 1.0 μ g/ μ l make 4 kinds of dna solutions with the 50mM carbonate buffer solution with concentration adjustment then.

5. negative control: will use λ and have a liking for thallus DNA as template with have pcr amplification product (300bp) subclone that the combination of the primer of sequence shown in sequence table SEQ ID NO:33 or 35 obtains and go in the pUC19 carrier.Use then and have the primer of sequence shown in sequence table SEQ ID NO:25 or 26 through pcr amplification subclone product.Thus obtained product as primer, is used for product by the about 400bp of amplification method amplification of the present invention with them, with the acquisition negative control as template and primer with sequence shown in sequence table SEQ ID NO:31 or 32.5 kinds of dna solutions are used for point sample, and described dna solution is an original content reaction mixture or with the reaction mixture of 2 times, 4 times, 8 times of carbonate buffer solution (using the carbonate buffer solution of 50mM concentration to dilute under each situation) dilutions or 16 times.

6. negative control: usefulness Microcon-100 (Takara Shuzo) handles above-mentioned 5 dna fragmentations that obtain.Be that 0.125 μ g/ μ l, 0.25 μ g/ μ l, 0.5 μ g/ μ l, 1.0 μ g/ μ l and 2.0 μ g/ μ l make 5 kinds of dna solutions with the 50mM carbonate buffer solution with concentration adjustment then.

The corresponding dna solution point sample of so preparation is added on the amino slide glass (Matsunami Glass) at the equipment (Genetic Microsystems (GMS)) of using preparation DNA chip, and utilize the UV radiation to fix.Slide glass washs with 0.2%SDS, uses distilled water wash then, and dry back is as the DNA array.

Use Label IT Cy5 ^RLabelling kit (Takara Shuzo) makes Cy5 on pcr amplification product (300bp) mark that the combination that utilizes the primer of sequence shown in sequence table SEQ ID NO:29 or 30 obtains, with as probe.According to the incidental specification sheets introduction of IntelliGene (Takara Shuzo), use a kind of prehybridization solution and a kind of hybridization solution is hybridized.At first, make DNA array prehybridization 2 hours under room temperature.The hybridization solution that will contain sex change Cy5 label probe drops on the DNA array.Covered thereon.Use the film phonograph seal cover glass.The DNA array of sealing was in 65 ℃ of incubations 13 hours.After removing cover glass, described DNA array, in 65 ℃ of washings 5 minutes, washed 5 minutes under room temperature in 0.2 * SSC in the solution that contains 0.2 * SSC and 0.1%SDS in 65 ℃ of washings 5 minutes at last with 2 * SSC, and is air-dry.Use the fluorescent signal of the corresponding spot of microarray scanner (GMS) analyzing DNA array then.

The result observes fluorescent signal at segmental each position of point sample PCR method (above-mentioned 3 and 4 described positive control) and the inventive method (above-mentioned 1 and 2 described sample) amplification.Strength of signal is as follows: sample 2＞positive control 4＞sample 1＞positive control 3.Otherwise, all do not observe signal at all sites of negative control 5 and 6.These results confirm, what the non-purifying of the inventive method amplification or purifying DNA fragment can be preferably used as preparation DNA chip treats the fixed dna fragment.

Embodiment 12

(1) studies the primer design that the fragment of pcr amplification is wherein used as the inventive method of template.At first, according to the synthetic primer of ordinary method with one of sequence shown in the sequence table SEQ ID NO:36-41.The structure of corresponding primer is as follows.

(i) R1-S1 primer: from 5 '-end is the intervening sequence of 7 bases, the M13RV sequence of 17 bases (or RV sequence; The nucleotide sequence of M13RV primer (Takara Shuzo)) and 20 bases adopted primer sequence arranged, be used for λ DNA specific PCR;

(ii) R1-A3 primer: from 5 '-end is the M13RV sequence of the intervening sequence of 7 bases, 17 bases and the antisense primer sequence of 20 bases, is used for λ DNA specific PCR;

(iii) R2-S1 primer: from 5 '-end is the adopted primer sequence that has of the M13RV sequence of the intervening sequence of 25 bases, 17 bases and 20 bases, is used for λ DNA specific PCR;

(iv) R2-A3 primer: from 5 '-end is the M13RV sequence of the intervening sequence of 25 bases, 17 bases and the antisense primer sequence of 20 bases, is used for λ DNA specific PCR;

(v) R3-S1 primer: from 5 '-end is the adopted primer sequence that has of the M13RV sequence of the intervening sequence of 58 bases, 17 bases and 20 bases, is used for λ DNA specific PCR; With

(vi) R3-A3 primer: from 5 '-end is the M13RV sequence of the intervening sequence of 58 bases, 17 bases and the antisense primer sequence of 20 bases, is used for λ DNA specific PCR.

M13RV 20mer has the sequence of 20 bases altogether of 3 based compositions of M13RV sequence and the 5 '-end of 17 bases.Therefore, when M13RV 20mer was used for the inventive method, the intervening sequence length of above-mentioned primer was respectively 4 bases, 22 bases and 55 bases.Also preparation does not have the contrast of the primer of intervening sequence as above-mentioned primer.

For example when using primer, obtain the amplified fragments of 348bp to R1-S1 primer/R1-A3 primer.7 bases at the amplified fragments two ends are equivalent to compartment.The RV sequence is positioned at the inside of compartment.The λ dna sequence dna is positioned at RV sequence inside.

Equally, when using primer, obtain the amplified fragments that 25 bases at amplified fragments two ends wherein are equivalent to the 384bp of compartment to R2-S1 primer/R2-A3 primer.In addition, when using primer, obtain the amplified fragments that 58 bases at amplified fragments two ends wherein are equivalent to the 450bp of compartment to R3-S1 primer/R3-A3 primer.Otherwise, use the amplified fragments of contrast primer not have compartment.These pcr amplified fragments are as the template that detects subsequently.

One of two kinds of primers are that M13RV-2N 17mer primer or M13RV-2N 20mer have sequence shown in sequence table SEQ ID NO:42 or 43, and they are used for present embodiment.In described primer, first of 3 '-end and second base are replaced by ribonucleotide.The following reaction.The mixture of the 20 μ M primers of 5 μ l, about 20ng template and 0.01% propylene diamine is cooled to 55 ℃ then in 98 ℃ of sex change 2 minutes.To wherein adding 34mM Tricine buffer reagent (pH 8.7), 10mM Repone K, 10mM ammonium sulfate, 0.01%BSA, 1%DMSO, 4mM magnesium acetate, each dNTP of 0.5mM, 1U Bca BEST archaeal dna polymerase and 15U ribonuclease H, make that the end reaction volume is 25 μ l.Make reaction mixture in 55 ℃ of incubations 1 hour.After reaction is finished, make mixture be cooled to 4 ℃, then to wherein adding 2.5 μ l 0.5M EDTA solution stopped reactions.Get 3 μ l reaction mixtures at 3%NuSieve3: carry out electrophoresis on 1 agarose (Takara Shuzo) gel.The result is when using M13RV-2N17mer, and observing amplification efficiency increases with the intervening sequence length relation as follows: 25mer＞7mer＞58mer＞and continuously every sequence.When using M13RV-2N 20mer, observing amplification efficiency increases with the relation of intervening sequence length as follows: 22mer＞4mer＞55mer＞and continuously every sequence.In addition, as above-mentioned (i)-(when the M13RV sequence in the primer is vi) replaced by the M13M4 sequence, observe the similar trend of intervening sequence and amplification efficiency relation.Therefore confirm that the primer that is used for the inventive method that design produces intervening sequence (part) makes the amplification efficiency raising when linear DNA fragment such as pcr amplified fragment during as template.

(2) research and utilization raising temperature of reaction has the template of high GC content with the method amplification of amplification of nucleotide acid sequence.At first, prepare primer, be used for 307-bp section (the GC content: 62.5%) of pcr amplification CDC2-related protein kinase PISS LRE gene (GenBank accession number AA789328) with sequence shown in sequence table SEQ ID NO:44 or 45.In addition, prepare primer, be used for 284-bp section (the GC content: 61.3%) of pcr amplification II type cytoskeleton 1 keratin gene (GenBank accession number AA706022) with sequence shown in sequence table SEQ ID NO:46 or 47.Utilize these primers and commercially available dna fragmentation (Research Genetics) to carry out pcr amplification as template.Use above-mentioned primer the corresponding pcr amplified fragment that obtains is had intervening sequence and M13RV sequence at two ends.Described fragment is as template of the present invention.

The M13RV-2N 20mer primer that has the M13RV-2N 17mer primer of sequence shown in the sequence table SEQ ID NO:42 or have a sequence shown in the sequence table SEQ ID NO:43 is used for present embodiment.In described primer, first of 3 '-end and second base are replaced by ribonucleotide.The following reaction.The mixture of the 100pM primer of 10 μ l, 20ng template and 0.01% propylene diamine is cooled to 55 ℃ or 60 ℃ then in 98 ℃ of sex change 2 minutes.To wherein adding 34mM Tricine buffer reagent (pH 8.7), 10mM Repone K, 10mM ammonium sulfate, 0.01%BSA, 1%DMSO, 4mM magnesium acetate, each dNTP of 0.5mM, 11U Bca BEST archaeal dna polymerase and 30U ribonuclease H, make that the end reaction volume is 50 μ l then.Make reaction mixture in 55 ℃ or 60 ℃ of incubations 1 hour.After reaction is finished, make mixture be cooled to 4 ℃.Get 3 μ l reaction mixtures and on 3% sepharose, carry out electrophoresis.The results are shown in following table 1.

Table 1

Temperature of reaction	The primer	Gene and amplification
		Gene and amplification		CDC2-is correlated with	II type cytoskeleton
		55℃	M13RV-2N 17mer M13RV-2N 20mer	CDC2-is correlated with	II type cytoskeleton	++ ++	++ ++
60℃	M13RV-2N 17mer M13RV-2N 20mer	55℃	M13RV-2N 17mer M13RV-2N 20mer	+ ++++	+ ++++	++ ++	++ ++

+ extremely ++ and ++: with 4 level evaluations amplification degree.

-: do not observe amplification.

As shown in table 1, have the template of high GC content even use, the purpose section also effectively increases.Followingly finish this amplification: rising temperature of reaction (being increased to 60 ℃) and when reacting on 60 ℃ when carrying out from 55 ℃, use primer with high Tm value (with respect to the suitableeest primer that reacts on 55 ℃).

(3) length and the relation between the amplified production amount of amplification of nucleotide sequence method under the high reaction temperature condition of research amplified fragments.At first, it is right to synthesize the primer with sequence shown in sequence table SEQ ID NO:48 or 49 according to ordinary method, the 800-bp section (Takara Shuzo) of λ DNA is used to increase, and the primer with sequence shown in sequence table SEQ ID NO:50 or 51 is right, the 400-bp section of the λ DNA that is used to increase.Adopt these two kinds of primers to one of carry out PCR with λ DNA as template, to obtain amplified fragments.Also adopt the amplified fragments of embodiment 5 (1) described pUC19-911 plasmids as template and MF2 (24) primer and the about 1.1kbp of MR1 (24) primer preparation, described primer has the sequence shown in sequence table SEQ ID NO:16 and 17 respectively.Use above-mentioned primer the fragment that obtains through pcr amplification is had intervening sequence and M13RV or MR sequence at two ends.These fragments are as template of the present invention.

Have the M13RV-2N 17mer primer of sequence shown in the sequence table SEQ ID NO:42 or have the primer of the M13RV-2N 20mer primer of sequence shown in the sequence table SEQ ID NO:43 as present embodiment.In described primer, first of 3 '-end and second base are replaced by ribonucleotide.M13M4-3N 20mer primer and combination with sequence shown in the sequence table SEQ ID NO:55 with M13RV-3N 20mer primer of sequence shown in the sequence table SEQ ID NO:43, and the combination that has the M13M4-3N 24mer primer and the M13RV-3N 24mer primer of sequence shown in sequence table SEQ ID NO:56 and 57 respectively, the section of about 1 kbp that is used to increase.In described primer, first of 3 '-end to the 3rd base replaced by ribonucleotide.The following reaction.The mixture that makes the 10pM primer of 10 μ l, about 20ng template and 0.01% propylene diamine is cooled to 55 ℃ or 60 ℃ then in 98 ℃ of sex change 2 minutes.After this to wherein adding 34mM Tricine buffer reagent (pH 8.7), 10mM Repone K, 10mM ammonium sulfate, 0.01%BSA, 1%DMSO, 4mM magnesium acetate, each dNTP of 0.5mM, 11U Bca BEST archaeal dna polymerase and 30U ribonuclease H, making the end reaction volume is 50 μ l.Make reaction mixture in 55 ℃ or 60 ℃ of incubations 1 hour.After finishing reaction, make mixture be cooled to 4 ℃, then to wherein adding 5 μ l 0.5M EDTA solution stopped reactions.Get 3 μ l reaction mixtures and on 3: 1 agaroses (Takara Shuzo) of 3%NuSieve gel, carry out electrophoresis.The results are shown in following table 2 and 3.

Table 2

Temperature of reaction	The primer	The length of amplified fragments and result
		The length of amplified fragments and result		400bp	800bp
		55℃	M13RV-2N 17mer M13RV-2N 20mer	400bp	800bp	++ ++	++ ++
60℃	M13RV-2N 17mer M13RV-2N 20mer	55℃	M13RV-2N 17mer M13RV-2N 20mer	+ ++++	++++	++ ++	++ ++

+ extremely ++ and ++: with 4 level evaluations amplification degree.

-: do not observe amplification.

As shown in table 2, by making amplimer length become 20mer and making temperature of reaction from 55 ℃ of fragments that are increased to 60 ℃ of effectively increase 400bp and 800bp sections by 17mer.

Table 3

Temperature of reaction	The primer	The length of amplified fragments and 1034bp as a result
Temperature of reaction	The primer	The length of amplified fragments and 1034bp as a result	55℃	M13RV-3N 20mer & M13M4-3N 20mer M13RV-3N 24mer & M13M4-3N 24mer	++ ++
65℃	M13RV-3N 20mer & M13M4-3N 20mer M13RV-3N 24mer & M13M4-3N 24mer	+ ++++	55℃		++ ++

+ extremely ++ and ++: with 4 level evaluations amplification degree.

-: do not observe amplification.

In addition, as shown in table 3, by making amplimer length be extended for 24mer and making temperature of reaction be increased to 65 ℃ of effectively fragments of the about 1kbp section of amplification from 55 ℃ by 20mer.In addition, utilize longer primer and temperature of reaction to raise, described the same with embodiment 10, to the similar result of amplification acquisition of long-chain dna fragmentation.When about 2kbp of amplification or longer section, observe the amplification efficiency raising.

Embodiment 13

(1) research hot resistant DNA polymerase rather than the application in the methods of the invention of Bca BEST archaeal dna polymerase.Bst archaeal dna polymerase (New England Biolabs) is as hot resistant DNA polymerase.According to synthetic a pair of primer 5 '-ID primer and the 3 '-ID primer that has sequence shown in sequence table SEQ ID NO:52 and 53 respectively of ordinary method.Use described primer the commercially available dna fragmentation (Research Genetics) with the A cyclin gene is carried out PCR as template, obtain the amplified fragments of about 300bp.Use described primer and have the M13RV sequence at two ends obtaining pcr amplified fragment.Described fragment is as template of the present invention.

Have the primer of the M13RV-2N 17mer primer of sequence shown in the sequence table SEQ ID NO:42 as present embodiment.In described primer, first of 3 '-end and second base are replaced by ribonucleotide.The following reaction.The mixture that makes the 20 μ M primers of 10 μ l, about 20ng template and 0.01% propylene diamine is cooled to 55 ℃ then in 98 ℃ of sex change 2 minutes.After this to wherein adding 34mM Tricine buffer reagent (pH 8.7), 10mM Repone K, 10mM ammonium sulfate, 0.01%BSA, 1%DMSO, 4mM magnesium acetate, each dNTP of 0.5mM, 4,8,12 or 16U Bst archaeal dna polymerase and 30U ribonuclease H, making the end reaction volume is 50 μ l.In contrast, preparation and the identical reaction mixture of above-mentioned reaction mixture composition, the different 11U Bca BEST archaeal dna polymerases that are to use.Make reaction mixture in 55 ℃ of incubations 1 hour.After finishing reaction, make mixture be cooled to 4 ℃, then to wherein adding 5 μ l 0.5M EDTA solution stopped reactions.Get 3 μ l reaction mixtures and on 3: 1 agaroses (Takara Shuzo) of 3%NuSieve gel, carry out electrophoresis.The Bst archaeal dna polymerase that the result uses each described not commensurability obtains the purpose amplified fragments.Therefore confirm that hot resistant DNA polymerase is preferred for method of the present invention.

(2) application in the methods of the invention of warm archaeal dna polymerase is had a liking in research.The Klenow fragment of 5 '-3 ' circumscribed activity (-) (Takara Shuzo) is as having a liking for warm archaeal dna polymerase.The DNA of above-mentioned (1) preparation is as the template DNA of the inventive method.

Have the primer of the M13RV-2N 16mer primer of sequence shown in the sequence table SEQ ID NO:54 as present embodiment.In described primer, first of 3 '-end and second base are replaced by ribonucleotide.The following reaction.The mixture that makes the 20 μ M primers of 10 μ l, about 20ng template and 0.01% propylene diamine is cooled to 40 ℃ then in 98 ℃ of sex change 2 minutes.After this to wherein adding 34mM Tricine buffer reagent (pH 8.7), 10mM Repone K, 10mM ammonium sulfate, 0.01%BSA, 1%DMSO, 4mM magnesium acetate, each dNTP of 0.5mM, 0,2,4,6 or 8U Klenow fragment and 30U ribonuclease H, making the end reaction volume is 50 μ l.Make reaction mixture in 40 ℃ of incubations 1 hour.After finishing reaction, make mixture be cooled to 4 ℃, then to wherein adding 5 μ l 0.5M EDTA solution stopped reactions.Get 3 μ l reaction mixtures and on 3: 1 agaroses (Takara Shuzo) of 3%NuSieve gel, carry out electrophoresis.The result when not adding the Klenow fragment, using not, the Klenow fragment of commensurability all obtains the purpose amplified fragments.Therefore confirm that having a liking for warm archaeal dna polymerase can be preferred for method of the present invention.

Embodiment 14

Research will be used for the chimeric oligonucleotide primer of the inventive method.As synthetic template DNA and primer as described in the embodiment 1 (1).The structure that is used for the primer of present embodiment is described in detail as follows:

Primer is to 2: wherein 3 '-and the 6th terminal and the 7th combination of primers that deoxyribonucleotide is replaced by ribonucleotide with nucleotide sequence shown in sequence table SEQ ID NO:59 or 60;

Primer is to 3: wherein 3 '-and the 5th terminal and the 6th combination of primers that deoxyribonucleotide is replaced by ribonucleotide with nucleotide sequence shown in sequence table SEQ ID NO:61 or 62;

Primer is to 4: wherein 3 '-and the 4th terminal and the 5th combination of primers that deoxyribonucleotide is replaced by ribonucleotide with nucleotide sequence shown in sequence table SEQ ID NO:63 or 64;

Primer is to 5: wherein 3 '-and the 3rd terminal and the 4th combination of primers that deoxyribonucleotide is replaced by ribonucleotide with nucleotide sequence shown in sequence table SEQ ID NO:65 or 66;

Primer is to 6: wherein 3 '-and second terminal and the 3rd combination of primers that deoxyribonucleotide is replaced by ribonucleotide with nucleotide sequence shown in sequence table SEQ ID NO:67 or 68;

Primer is to 7: wherein 3 '-and terminal first and second combination of primers that deoxyribonucleotide is replaced by ribonucleotide with nucleotide sequence shown in sequence table SEQ ID NO:2 or 3; And

Primer is to 8: wherein 3 '-terminal second and the 3rd deoxyribonucleotide replaced by ribonucleotide and the phosphate bond of 5 '-end side of 3 '-terminal the 3rd ribonucleotide by the replacement of thiophosphoric acid key, have the combination of primers of nucleotide sequence shown in sequence table SEQ ID NO:67 or 68.

Amplification condition and detection method are as described in embodiment 1 (2) and (3).Each primer observes the amplified fragments with purpose length to 2-8 as a result.To 2-7, the amplified production amount increases with the minimizing of the deoxyribonucleotide number of 3 '-end for primer.The primer that does not have deoxyribonucleotide at 3 '-end is to 7 amplified productions that observe maximum amount.Especially it is maximum to 7 amplified production to observe the primer that do not have a deoxyribonucleotide at 3 '-end.Otherwise, do not observe amplified fragments to 1 at primer.In addition, at primer 6 and 8 the two fact that all observe the purpose amplified fragments are confirmed that the two all can be preferably used as the ribonucleotide that the primer in the inventive method contains to modify ribonucleotide and non-modification ribonucleotide.

Industrial usability

The invention provides the conventional effective ways of amplification of nucleotide acid sequence, it is characterized in that carrying out synthesis reaction of DNA under the chimeric oligonucleotide primer existing. The invention provides the method for a large amount of supply amplification of DNA fragments. Also by method and the another kind of nucleic acid amplification method of associating amplification of nucleotide acid sequence of the present invention, provide the effective ways of amplification of nucleotide acid sequence. In addition, the invention provides for detection of or quantitatively microorganism such as the method for the detection nucleotide sequence of virus, bacterium, fungi and yeast and the method that detects in real time the amplification of DNA fragments of the inventive method acquisition. In addition, the invention provides the method for extensive gene sequencing.

Sequence table independence text

SEQ ID NO:1: the synthetic DNA that is used as the part that is equivalent to the human transferrin receptor coding sequence of template.

SEQ ID NO:2: the Oligonucleolide primers that is designed for the part of amplification human transferrin receptor coding sequence.

SEQ ID NO:3: the Oligonucleolide primers that is designed for the part of amplification human transferrin receptor coding sequence.

SEQ ID NO:4: design is as the oligonucleotide of the probe of the amplification part that detects the human transferrin receptor coding sequence.

SEQ ID NO:5: be designed for the Oligonucleolide primers of the part of amplification plasmid pUC19, be called pUC19 upstream (2) NN.

SEQ ID NO:6: be designed for the Oligonucleolide primers of the part of amplification plasmid pUC19, be called pUC19 downstream NN.

SEQ ID NO:7: the Oligonucleolide primers that is designed for the part of amplification plasmid pUC19.

SEQ ID NO:8: be designed for the Oligonucleolide primers of the part of amplification plasmid pUC19, be called pUC19 downstream 542.

SEQ ID NO:9: the Oligonucleolide primers that is designed for the part of amplification plasmid pUC19.

SEQ ID NO:10: be designed for the Oligonucleolide primers of the part of amplification plasmid pUC19, be called pUC19 upstream 150.

SEQ ID NO:11: be designed for the Oligonucleolide primers of the part of amplification plasmid pUC19, be called pUC19 upstream 249.

SEQ ID NO:12: the Oligonucleolide primers that is designed for the part of amplification human transferrin receptor coding sequence.

SEQ ID NO:13: the Oligonucleolide primers that is designed for the part of amplification human transferrin receptor coding sequence.

SEQ ID NO:14: the Oligonucleolide primers that is designed for the part of amplification human transferrin receptor coding sequence.

SEQ ID NO:15: the Oligonucleolide primers that is designed for the part of amplification human transferrin receptor coding sequence.

SEQ ID NO:16: be designed for the Oligonucleolide primers of the part of amplification plasmid pUC19-249 or plasmid pUC19-911, be called MF2N3 (24).

SEQ ID NO:17: be designed for the Oligonucleolide primers of the part of amplification plasmid pUC19-249 or plasmid pUC19-911, be called MR1N3 (24).

SEQ ID NO:18: the Oligonucleolide primers that is designed for the part of amplification plasmid pUC19.

SEQ ID NO:19: be designed for the Oligonucleolide primers of the part of amplification plasmid pUC19, be called MR1N3.

SEQ ID NO:20: as the synthetic RNA of the amplification that detects plasmid pUC19 probe partly.

SEQ ID NO:21: the Oligonucleolide primers that is designed for the part of Vero cytotoxin 1 encoding sequence that amplifies blood group intestinal bacteria O-157.

SEQ ID NO:22: the Oligonucleolide primers that is designed for the part of Vero cytotoxin 1 encoding sequence that amplifies blood group intestinal bacteria O-157.

SEQ ID NO:23: the Oligonucleolide primers that is designed for the part of Vero cytotoxin 2 encoding sequences that amplify blood group intestinal bacteria O-157.

SEQ ID NO:24: the Oligonucleolide primers that is designed for the part of Vero cytotoxin 2 encoding sequences that amplify blood group intestinal bacteria O-157.

SEQ ID NO:25: be designed for the segmental Oligonucleolide primers of amplification length dna, be called MCR-F.

SEQ ID NO:26: be designed for the segmental Oligonucleolide primers of amplification length dna, be called MCR-R.

SEQ ID NO:27: be designed for the segmental Oligonucleolide primers of amplification length dna, be called MF2N3 (24).

SEQ ID NO:28: be designed for the segmental Oligonucleolide primers of amplification length dna, be called MR1N3 (24).

SEQ ID NO:29: be designed for the Oligonucleolide primers that amplification λ has a liking for the part of thallus DNA.

SEQ ID NO:30: be designed for the Oligonucleolide primers that amplification λ has a liking for the part of thallus DNA.

SEQ ID NO:31: be designed for the Oligonucleolide primers that amplification λ has a liking for the part of thallus DNA.

SEQ ID NO:32: be designed for the Oligonucleolide primers that amplification λ has a liking for the part of thallus DNA.

SEQ ID NO:33: be designed for the Oligonucleolide primers that amplification λ has a liking for the part of thallus DNA.

SEQ ID NO:34: be designed for the Oligonucleolide primers that amplification λ has a liking for the part of thallus DNA.

SEQ ID NO:35: be designed for the Oligonucleolide primers that amplification λ has a liking for the part of thallus DNA.

SEQ ID NO:36: be designed for the Oligonucleolide primers that amplification λ has a liking for the part of thallus DNA, be called R1-S1.

SEQ ID NO:37: be designed for the Oligonucleolide primers that amplification λ has a liking for the part of thallus DNA, be called R1-A3.

SEQ ID NO:38: be designed for the Oligonucleolide primers that amplification λ has a liking for the part of thallus DNA, be called R2-S1.

SEQ ID NO:39: be designed for the Oligonucleolide primers that amplification λ has a liking for the part of thallus DNA, be called R2-A3.

SEQ ID NO:40: be designed for the Oligonucleolide primers that amplification λ has a liking for the part of thallus DNA, be called R3-S1.

SEQ ID NO:41: be designed for the Oligonucleolide primers that amplification λ has a liking for the part of thallus DNA, be called R3-A3.

SEQ ID NO:42: the design oligonucleotides primer that is called M13RV-2N 17mer.

SEQ ID NO:43: the design oligonucleotides primer that is called M13RV-2N 20mer.

SEQ ID NO:44: the Oligonucleolide primers that is designed for the part of amplification CDC2 related protein kinase PISSLRE gene.

SEQ ID NO:45: the Oligonucleolide primers that is designed for the part of amplification CDC2 related protein kinase PISSLRE gene.

SEQ ID NO:46: the Oligonucleolide primers that is designed for the part of amplification II type cytoskeleton 11 keratin genes.

SEQ ID NO:47: the Oligonucleolide primers that is designed for the part of amplification II type cytoskeleton 11 keratin genes.

SEQ ID NO:48: be designed for the Oligonucleolide primers that amplification λ has a liking for the part of thallus DNA.

SEQ ID NO:49: be designed for the Oligonucleolide primers that amplification λ has a liking for the part of thallus DNA.

SEQ ID NO:50: be designed for the Oligonucleolide primers that amplification λ has a liking for the part of thallus DNA.

SEQ ID NO:51: be designed for the Oligonucleolide primers that amplification λ has a liking for the part of thallus DNA.

SEQ ID NO:52: be designed for the Oligonucleolide primers of the part of amplification A cyclin DNA, be called 5 ' ID.

SEQ ID NO:53: be designed for the Oligonucleolide primers of the part of amplification A cyclin DNA, be called 3 ' ID.

SEQ ID NO:54: the design oligonucleotides primer that is called M13RV-2N 16mer.

SEQ ID NO:55: the design oligonucleotides primer that is called M13M4-3N 16mer.

SEQ ID NO:56: the design oligonucleotides primer that is called M13M4-3N 24mer.

SEQ ID NO:57: the design oligonucleotides primer that is called M13RV-3N 24mer.

SEQ ID NO:58: the design oligonucleotides primer that is called M13M4 17mer.

SEQ ID NO:59: the Oligonucleolide primers that is designed for the part of amplification human transferrin receptor coding sequence.

SEQ ID NO:60: the Oligonucleolide primers that is designed for the part of amplification human transferrin receptor coding sequence.

SEQ ID NO:61: the Oligonucleolide primers that is designed for the part of amplification human transferrin receptor coding sequence.

SEQ ID NO:62: the Oligonucleolide primers that is designed for the part of amplification human transferrin receptor coding sequence.

SEQ ID NO:63: the Oligonucleolide primers that is designed for the part of amplification human transferrin receptor coding sequence.

SEQ ID NO:64: the Oligonucleolide primers that is designed for the part of amplification human transferrin receptor coding sequence.

SEQ ID NO:65: the Oligonucleolide primers that is designed for the part of amplification human transferrin receptor coding sequence.

SEQ ID NO:66: the Oligonucleolide primers that is designed for the part of amplification human transferrin receptor coding sequence.

SEQ ID NO:67: the Oligonucleolide primers that is designed for the part of amplification human transferrin receptor coding sequence.

SEQ ID NO:68: the Oligonucleolide primers that is designed for the part of amplification human transferrin receptor coding sequence.

Sequence table

<110>Takara Shuzo Co..Ltd.

＜120〉be used for the method for amplification of nucleotide acid sequence

<130>661724

<150>JP 11-076966

<151>1999-03-19

<150>JP 11-370035

<151>1999-12-27

<160>68

<210>1

<211>99

<212>DNA

＜213〉artificial sequence

<220>

＜223〉as the synthetic DNA of human transferrin receptor coding sequence that is equivalent to a part of template

<400>1

ggacagcaac tgggccagca aagttgagaa actcacttta gagaattctg ctttcccttt 60

ccttgcatat tctgagcagt ttctttctgt ttttgcgag 99

<210>2

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be designed for the Oligonucleolide primers of the human transferrin receptor coding sequence of an amplification part

<400>2

cagcaactgg gccagcaaag tt 22

<210>3

<211>22

<212>DNA

＜213〉artificial sequence

<220>

<400>3

gcaaaaacag aaagaaactg ct 22

<210>4

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉design is as the oligonucleotide of the probe of the amplification part that detects the human transferrin receptor coding sequence

<400>4

tgctttccct ttccttgcat attctg 26

<210>5

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be designed for the Oligonucleolide primers of the part of amplification plasmid pUC19, be called pUC19 upstream (2) NN

<400>5

attgcttaat cagtgaggca cctat 25

<210>6

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be designed for the Oligonucleolide primers of the part of amplification plasmid pUC19, be called pUC19 downstream NN

<400>6

gataacactg cggccaactt acttc 25

<210>7

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be designed for the Oligonucleolide primers of the part of amplification plasmid pUC19

<400>7

actggcgaac tacttactct agctt 25

<210>8

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be designed for the Oligonucleolide primers of the part of amplification plasmid pUC19, be called pUC19 downstream 542

<400>8

agtcaccaga aaagcatctt acggat 26

<210>9

<211>25

<212>DNA

＜213〉artificial sequence

<220>

<400>9

gctcatgaga caataaccct gataa 25

<210>10

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be designed for the Oligonucleolide primers of the part of amplification plasmid pUC19, be called pUC19 upstream 150

<400>10

ggtgtcacgc tcgtcgtttg gtatg 25

<210>11

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be designed for the Oligonucleolide primers of the part of amplification plasmid pUC19, be called pUC19 upstream 249

<400>11

cgcctccatc cagtctatta attgt 25

<210>12

<211>22

<212>DNA

＜213〉artificial sequence

<220>

<400>12

ctgattgaga ggattcctga gt 22

<210>13

<211>22

<212>DNA

＜213〉artificial sequence

<220>

<400>13

tagggagaga ggaagtgata ct 22

<210>14

<211>22

<212>DNA

＜213〉artificial sequence

<220>

<400>14

caacttcaag gtttctgcca gc 22

<210>15

<211>21

<212>DNA

＜213〉artificial sequence

<220>

<400>15

aatagtccaa gtagctagag c 21

<210>16

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be designed for the Oligonucleolide primers of the part of amplification plasmid pUC19-249 or plasmid pUC19-911,

Be called MF2N3 (24)

<400>16

gctgcaaggc gattaagttg ggta 24

<210>17

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be designed for the Oligonucleolide primers of amplification plasmid pUC19-249 or plasmid pUC19-911, be called

MR1N3(24)

<400>17

ctttatgctt ccggctcgta tgtt 24

<210>18

<211>30

<212>DNA

＜213〉artificial sequence

<220>

<400>18

ggatgtgctg caaggcgatt aagttgggta 30

<210>19

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be designed for the Oligonucleolide primers of the part of amplification plasmid pUC19, be called MR1N3

<400>19

tttacacttt atgcttccgg ctcgtatgtt 30

<210>20

<211>30

<212>RNA

＜213〉artificial sequence

<220>

＜223〉as the synthetic RNA of the probe of the amplification part that detects plasmid pUC19

<400>20

ugauccccca uguugugcaa aaaagcgguu 30

<210>21

<211>18

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be designed for the part of Vero cytotoxin 1 encoding sequence that amplifies blood group intestinal bacteria 0-157

Oligonucleolide primers

<400>21

agttaatgtg gtggcgaa 18

<210>22

<211>17

<212>DNA

＜213〉artificial sequence

<220>

Oligonucleolide primers

<400>22

gactcttcca tctgcca 17

<210>23

<211>18

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be designed for the part of Vero cytotoxin 2 encoding sequences that amplify blood group intestinal bacteria 0-157

Oligonucleolide primers

<400>23

ttcggtatcc tattcccg 18

<210>24

<211>18

<212>DNA

＜213〉artificial sequence

<220>

Oligonucleolide primers

<400>24

tctctggtca ttgtatta 18

<210>25

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be designed for the segmental Oligonucleolide primers of amplification length dna, be called MCR-F

<400>25

ccattcaggc tgcgcaactg tt 22

<210>26

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be designed for the segmental Oligonucleolide primers of amplification length dna, be called MCR-R

<400>26

tggcacgaca ggtttcccga ct 22

<210>27

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be designed for the segmental Oligonucleolide primers of amplification length dna, be called MF2N3 (24)

<400>27

gctgcaaggc gattaagttg ggta 24

<210>28

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be designed for the segmental Oligonucleolide primers of amplification length dna, be called MR1N3 (24)

<400>28

ctttatgctt ccggctcgta tgtt 24

<210>29

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be designed for the Oligonucleolide primers of a part of amplification lambda bacteriophage dna

<400>29

aacaacaaga aactggtttc 20

<210>30

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<400>30

gcaatgcatg acgactgggg 20

<210>31

<211>17

<212>DNA

＜213〉artificial sequence

<220>

<400>31

gttttcccag tcacgac 17

<210>32

<211>17

<212>DNA

＜213〉artificial sequence

<220>

<400>32

caggaaacag ctatgac 17

<210>33

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<400>33

gtacggtcat catctgacac 20

<210>34

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<400>34

gcaatcggca tgttaaacgc 20

<210>35

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<400>35

cgccatcctg ggaagactcc 20

<210>36

<211>44

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be designed for the Oligonucleolide primers of a part of amplification lambda bacteriophage dna, be called R1-S1

<400>36

tttcacacag gaaacagcta tgacaacaac aagaaactgg tttc 44

<210>37

<211>44

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be designed for the Oligonucleolide primers of a part of amplification lambda bacteriophage dna, be called R1-A3

<400>37

tttcacacag gaaacagcta tgacgcaatg catgacgact gggg 44

<210>38

<211>62

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be designed for the Oligonucleolide primers of a part of amplification lambda bacteriophage dna, be called R2-S1

<400>38

attgtgagcg gataacaatt tcacacagga aacagctatg acaacaacaa gaaactggtt 60

tc 62

<210>39

<211>62

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be designed for the Oligonucleolide primers of a part of amplification lambda bacteriophage dna, be called R2-A3

<400>39

attgtgagcg gataacaatt tcacacagga aacagctatg acgcaatgca tgacgactgg 60

gg 62

<210>40

<211>95

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be designed for the Oligonucleolide primers of a part of amplification lambda bacteriophage dna, be called R3-S1

<400>40

cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca 60

ggaaacagct atgacaacaa caagaaactg gtttc 95

<210>41

<211>95

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be designed for the Oligonucleolide primers of a part of amplification lambda bacteriophage dna, be called R3-A3

<400>41

cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca 60

ggaaacagct atgacgcaat gcatgacgac tgggg 95

<210>42

<211>17

<212>DNA

＜213〉artificial sequence

<220>

＜223〉She Ji Oligonucleolide primers is called M13RV-2N 17mer

<400>42

caggaaacag ctatgac 17

<210>43

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉She Ji Oligonucleolide primers is called M13RV-2N 20mer

<400>43

acacaggaaa cagc tatgac 20

<210>44

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be designed for the Oligonucleolide primers of the part of amplification CDC2 related protein kinase PISSLRE gene

<400>44

gagttcgtgt ccgtacaact atttcacaca ggaaacagct atgacccaac aagagcctat 60

agcttcgctc 70

<210>45

<211>67

<212>DNA

＜213〉artificial sequence

<220>

<400>45

tcgaaatcag ccacagcgcc atttcacaca ggaaacagct atgacccgct gtctttgagt 60

tgtggtg 67

<210>46

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be designed for the Oligonucleolide primers of the part of amplification II type cytoskeleton Keratin sulfate 11 genes

<400>46

gagttcgtgt ccgtacaact atttcacaca ggaaacagct atgacgctat tctgacatca 60

ctttccagac 70

<210>47

<211>66

<212>DNA

＜213〉artificial sequence

<220>

<400>47

tcgaaatcag ccacagcgcc atttcacaca ggaaacagct atgacgaatt ccactggtgg 60

cagtag 66

<210>48

<211>62

<212>DNA

＜213〉artificial sequence

<220>

<400>48

attgtgagcg gataacaatt tcacacagga aacagctatg acgtacggtc atcatctgac 60

ac 62

<210>49

<211>62

<212>DNA

＜213〉artificial sequence

<220>

<400>49

attgtgagcg gataacaatt tcacacagga aacagctatg acatgcgccg cctgaaccac 60

ca 62

<210>50

<211>62

<212>DNA

＜213〉artificial sequence

<220>

<400>50

attgtgagcg gataacaatt tcacacagga aacagctatg acctgctctg ccgcttcacg 60

ca 62

<210>51

<211>62

<212>DNA

＜213〉artificial sequence

<220>

<400>51

attgtgagcg gataacaatt tcacacagga aacagctatg acgcaatcgg catgttaaac 60

gg 62

<210>52

<211>69

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be designed for the Oligonucleolide primers of the part of amplification A cyclin DNA, be called 5 ' ID

<400>52

tcgaaatcag ccacagcgcc atttcacaca ggaaacagct atgacatgtt ttgggagaat 60

taagtctga 69

<210>53

<211>69

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be designed for the Oligonucleolide primers of the part of amplification A cyclin DNA, be called 3 ' ID

<400>53

gagttcgtgc cgtacaacta tttcacacag gaaacagcta tgacttacag atttagtgtc 60

tctggtggg 69

<210>54

<211>16

<212>DNA

＜213〉artificial sequence

<220>

＜223〉She Ji Oligonucleolide primers is called M13RV-2N 16mer

<400>54

aggaaacagc tatgac 16

<210>55

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉She Ji Oligonucleolide primers is called M13M4-3N 16mer

<400>55

agggttttcc cagtcacgac 20

<210>56

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉She Ji Oligonucleolide primers is called M13M4-3N 24mer

<400>56

cgccagggtt ttcccagtca cgac 24

<210>57

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉She Ji Oligonucleolide primers is called M13RV-3N 24mer

<400>57

tttcacacag gaaacagcta tgac 24

<210>58

<211>17

<212>DNA

＜213〉artificial sequence

<220>

＜223〉She Ji Oligonucleolide primers is called M13M4

<400>58

gttttcccag tcacgac 17

<210>59

<211>27

<212>DNA

＜213〉artificial sequence

<220>

<400>59

cagcaactgg gccagcaaag ttgagaa 27

<210>60

<211>27

<212>DNA

＜213〉artificial sequence

<220>

<400>60

gcaaaaacag aaagaaactg ctcagaa 27

<210>61

<211>26

<212>DNA

＜213〉artificial sequence

<220>

<400>61

cagcaactgg gccagcaaag ttgaga 26

<210>62

<211>26

<212>DNA

＜213〉artificial sequence

<220>

<400>62

gcaaaaacag aaagaaactg ctcaga 26

<210>63

<211>25

<212>DNA

＜213〉artificial sequence

<220>

<400>63

cagcaactgg gccagcaaag ttgag 25

<210>64

<211>25

<212>DNA

＜213〉artificial sequence

<220>

<400>64

gcaaaaacag aaagaaactg ctcag 25

<210>65

<211>24

<212>DNA

＜213〉artificial sequence

<220>

<400>65

cagcaactgg gccagcaaag ttga 24

<210>66

<211>24

<212>DNA

＜213〉artificial sequence

<220>

<400>66

gcaaaaacag aaagaaactg ctca 24

<210>67

<211>23

<212>DNA

＜213〉artificial sequence

<220>

<400>67

cagcaactgg gccagcaaag ttg 23

<210>68

<211>23

<212>DNA

＜213〉artificial sequence

<220>

<400>68

gcaaaaacag aaagaaactg ctc 23

Claims

1. test kit, it is used for the method for amplification of nucleotide acid sequence or mass production nucleic acid or is used for the method for the target nucleic acid of test sample, and described test kit is packaged form and comprises:

(1) has the active archaeal dna polymerase of strand displacement;

(2) endonuclease; With

(3) be used for the buffer reagent of strand replacement reaction;

The method that wherein is used for amplification of nucleotide acid sequence or mass production nucleic acid comprises step (A) and (B):

(A) by hybrid template nucleic acid, triphosphate deoxyribose nucleotide, have an endonuclease preparation feedback mixture of the extended chain that the active archaeal dna polymerase of strand displacement, at least a primer and cutting produce by primer, wherein said primer is for basic complementary and comprise the chimeric oligonucleotide primer of deoxyribonucleotide and ribonucleotide with the nucleotide sequence of described template nucleic acid, and described ribonucleotide is positioned on 3 ' of described primer-end or 3 '-end side; And

(B) can be annealed on the template nucleic acid specifically at described primer, take place under the condition of the strand replacement reaction of extended chain building-up reactions and described archaeal dna polymerase and the reaction that endonuclease cuts extended chain, the enough time of incubation reaction mixture is to produce reaction product;

The method that wherein is used for the target nucleic acid of test sample comprises step (i) and (ii):

(i) use the method amplifying target nucleic acid that comprises step (A) and amplification of nucleotide acid sequence (B) or mass production nucleic acid; And

(ii) detect the target nucleic acid of step (i) amplification.

2. the test kit of claim 1, it comprises that also guidance uses the specification sheets of archaeal dna polymerase and endonuclease in strand replacement reaction.

3. the test kit of claim 1, it comprises and is selected from following archaeal dna polymerase as described archaeal dna polymerase: the Bca archaeal dna polymerase of the Bst archaeal dna polymerase of the Klenow fragment of e. coli dna polymerase I, the shortage 5 ' of bacstearothermophilus → 3 ' exonuclease and shortage 5 ' → 3 ' exonuclease of hot hard genus bacillus.

4. the test kit of claim 1, it comprises the ribonuclease H as described endonuclease.

5. composition, it is used for the method for amplification of nucleotide acid sequence or mass production nucleic acid or is used for the method for the target nucleic acid of test sample, and described composition contains:

(1) has the active archaeal dna polymerase of strand displacement;

(2) endonuclease; With

(3) be used for the buffer reagent of strand replacement reaction;

(ii) detect the target nucleic acid of step (i) amplification.

6. the composition of claim 5, it comprises and is selected from following archaeal dna polymerase as described archaeal dna polymerase: the Bca archaeal dna polymerase of the Bst archaeal dna polymerase of the Klenow fragment of e. coli dna polymerase I, the shortage 5 ' of bacstearothermophilus → 3 ' exonuclease and shortage 5 ' → 3 ' exonuclease of hot hard genus bacillus.

7. the test kit of claim 5, it comprises the ribonuclease H as described endonuclease.

8. material with fixed nucleic acid, wherein said nucleic acid is arranged in the prospective region, and described material is to prepare with may further comprise the steps (i) and method (ii):

(i) fixed nucleic acid is treated in the method amplification of using amplification of nucleotide acid sequence or mass production nucleic acid; And

(ii) make the nucleic acid of step (i) amplification arrange and be fixed in the prospective region of upholder;

(B) can be annealed on the template nucleic acid specifically at described primer, take place under the condition of the strand replacement reaction of extended chain building-up reactions and described archaeal dna polymerase and the reaction that endonuclease cuts extended chain, the enough time of incubation reaction mixture is to produce reaction product.

9. the material of claim 8, wherein single-chain nucleic acid is arranged and is fixed in the described prospective region.

10. the material of claim 9, the single-chain nucleic acid that wherein is substantially free of its complementary strand is arranged and is fixed in the described prospective region.