CN102703400A - Hot start DNA (Deoxyribose Nucleic Acid) polymerase and application thereof - Google Patents
Hot start DNA (Deoxyribose Nucleic Acid) polymerase and application thereof Download PDFInfo
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- CN102703400A CN102703400A CN2012101858148A CN201210185814A CN102703400A CN 102703400 A CN102703400 A CN 102703400A CN 2012101858148 A CN2012101858148 A CN 2012101858148A CN 201210185814 A CN201210185814 A CN 201210185814A CN 102703400 A CN102703400 A CN 102703400A
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Abstract
The invention discloses a hot start DNA (Deoxyribose Nucleic Acid) polymerase and an application thereof. The polymerase comprises a single-chain DNA binding protein which can be combined with a primer, a double-chain DNA binding protein which can be combined with a DNA template, and a DNA polymerase. According to the hot start DNA polymerase disclosed by the invention, PCR (Polymerase Chain Reaction) specificity and sensitivity can be greatly improved.
Description
Technical field
The present invention relates to a kind of warm start archaeal dna polymerase and application thereof, be specifically related to contain two kinds of protein-bonded warm start archaeal dna polymerases of DNA, belong to biological technical field.
Background technology
PCR has been widely used in the molecular biology every field, and basic step comprises, sex change-annealing---extend.Specifically: the 1. sex change of template DNA: template DNA dissociates template DNA double-stranded DNA double-stranded or that form through pcr amplification after being heated to 94 ℃ of left and right sides certain hours, makes it to become strand, so that it combines with primer, for the lower whorl reaction is prepared.2. the annealing (renaturation) of template DNA and primer: template DNA is after heat denatured becomes strand, and temperature is reduced to the primer annealing temperature, and primer combines with the complementary sequence pairing of template DNA strand.3. the extension of primer: dna profiling--the primer binding substances is under the effect of high temperature-resisting DNA polymerase; With dNTP is reaction raw materials, and target sequence is a template, by base pairing and semiconservative replication principle; Synthetic new and a template DNA chain complementary semiconservative replication chain; Three processes are extended in recirculation sex change--annealing--, just can obtain more " semiconservative replication chain ", and this new chain can become next round-robin template again.Amplify millions of times through just waiting to expand the goal gene amplification after tens circulations.
Heat start PCR is to improve the amplification ability, specific amplification, the best a kind of method of amplification sensitivity.Traditional hot starts round pcr and mainly utilizes two kinds of methods: antibody sealing method and chemical sealing method.Two kinds of methods utilize chemical substance and antibody to combine with the dna polymerase activity center at normal temperatures to live with sealase respectively, and chemical substance or antibody inactivation under hot conditions discharge enzyme and live, thereby realize warm start.Antibody sealing method is through antibodies specific sealase active site, and this method enzyme belongs to non-bonding with antibodies and combines, and in enzyme preservation process, is prone to the risk that antibody activity reduces, and enzyme active center is not unique, is difficult to the complete closed enzyme and lives.Chemistry sealing method is to form stable amido linkage through amino on chemical substance and the high temperature-resisting DNA polymerase peptide chain, but complete closed enzymic activity at normal temperatures, and with the amido linkage hydrolysis, it is alive to discharge enzyme through high temperature.This method needs long-time high temperature (at least 15 minutes), the hydrolysis amido linkage, and long-time high temperature makes dna polymerase activity reduce easily; For the trace template, long-time high temperature is prone to make the template degraded.These two kinds of methods are not to realize the optimal method of warm start.
Summary of the invention
First technical problem to be solved by this invention provides a kind of warm start archaeal dna polymerase, and this enzyme has greatly improved PCR atopic, sensitivity.
Second technical problem to be solved by this invention provides the application of a kind of warm start archaeal dna polymerase in the heat start PCR reaction.
For solving the problems of the technologies described above, the present invention adopts following technical scheme to be:
The invention provides a kind of warm start archaeal dna polymerase, this enzyme comprises:
Can with primer bonded single-stranded DNA binding protein;
Can be conjugated protein with dna profiling bonded double-stranded DNA; And
Archaeal dna polymerase.
In warm start archaeal dna polymerase according to the invention, combining of two kinds of conjugated protein and dna sequence dnas is non-specific combination.Single-stranded DNA binding protein combines with the primer of single-stranded structure in the PCR system, combines with the dna profiling of duplex structure and double-stranded DNA is conjugated protein.Primer after conjugated protein and template can't form mixture, can not combine with archaeal dna polymerase, therefore can't be utilized by archaeal dna polymerase, thereby avoid producing non-specific amplification.Along with the carrying out of reaction, two kinds of conjugated proteins receive heat inactivation at preparatory denaturing step, discharge primer, template participation PCR reaction, and the PCR reaction is normally carried out, and realize warm start.
Further; " can with primer bonded single-stranded DNA binding protein " of the present invention; " can be conjugated protein with dna profiling bonded double-stranded DNA " can be that any single-stranded DNA binding protein or the double-stranded DNA with this function reported in the prior art are conjugated protein, and it can obtain through buying or preparing according to the method for prior art.
Further, said single-stranded DNA binding protein, double-stranded DNA conjugated protein with mol ratio archaeal dna polymerase be 1-2: 1-2: 10-15.
In order to specify technique effect of the present invention, the present invention provides a kind of single-stranded DNA binding protein at this, and its aminoacid sequence and provides a kind of double-stranded DNA conjugated protein shown in SEQ ID NO:1, and its aminoacid sequence is shown in SEQ ID NO:2.
Further; In order to reach better technique effect; Said archaeal dna polymerase is preferably the Taq archaeal dna polymerase; Can be through buying or the prior art for preparing acquisition; After for example Easy Taq DNA Polymerase (Beijing Quanshijin Biotechnology Co., Ltd), TaKaRa Taq (precious biotechnology Dalian ltd) or Taq DNA Polymerase (day root biochemical technology ltd) or all have the intestinal bacteria abduction delivering of Thermu aquaticus DNA polymerase gene from the clone, through the recombinant protein of the isolated 94KD of protein purification etc.
The present invention also provides the application of above-mentioned warm start archaeal dna polymerase in the heat start PCR reaction.
Advantage of the present invention is: traditional hot start method, need utilize chemical sealing method or antibody sealing method.Because these two kinds of methods are that archaeal dna polymerase is sealed, can't live by the complete closed enzyme, make hot start effect be affected.Method of the present invention is acted in a diametrically opposite way, and utilizes the substrate of two kinds of conjugated protein sealing archaeal dna polymerases of DNA: i.e. primer and template, and make sealing efficient can reach 100%, realize warm start more completely.Compare with traditional method, provided by the inventionly utilize the warm start archaeal dna polymerase to carry out the heat start PCR method to have higher amplification efficiency and specificity, the experiment that especially requires for highly sensitive, high specific like quantitative fluorescent PCR, has remarkable advantages.
Below in conjunction with accompanying drawing and embodiment the present invention is described further.
Description of drawings
Fig. 1: use the inventive method to realize the synoptic diagram of warm start;
Fig. 2: warm start archaeal dna polymerase and Platinum Taq enzymatic amplification comparative electrophoresis figure;
Wherein: swimming lane M:Trans2K Plus DNA Marker,
Swimming lane 1:Platinum Taq enzymatic amplification product
Swimming lane 2: warm start archaeal dna polymerase amplified production
Embodiment
Material and source used among the embodiment are respectively: Platinum Taq (American I nvitrogen company), Taq DNA Polymerase (day root biochemical technology ltd); EasyPure Genomic DNA Kit (Beijing Quanshijin Biotechnology Co., Ltd); Trans2K Plus DNA Marker (Beijing Quanshijin Biotechnology Co., Ltd); PEASY-E1 expression vector (Beijing Quanshijin Biotechnology Co., Ltd); E. coli bl21 (DE3) (full Shi Jin Bioisystech Co., Ltd), primer synthesizes (the handsome Bioisystech Co., Ltd in Beijing).
1 two kinds of protein-bonded acquisitions of DNA of embodiment
Two kinds of DNA of the present invention conjugated protein (single-stranded DNA binding protein and double-stranded DNA are conjugated protein) can be in the prior art disclosed any have combine the conjugated protein of dna single chain structure or duplex structure.For better explanation effect of the present invention, present embodiment provides two kinds, and to be present among the phage RB6 DNA conjugated protein.
According to RB6 genomic dna sequence (Enterobacteria phage RB69 (Bacteriophage RB69) DNA polymerase (Gp43) GenBank:U34036.1.) difference synthesizing single-stranded DNA protein-bonded nucleotide sequence SEQ ID NO:3 and the protein-bonded nucleotide sequence SEQ of double-stranded DNA ID NO:4 (Shanghai Sangon Biological Engineering Technology And Service Co., Ltd); The correct back of order-checking (the handsome Bioisystech Co., Ltd in Beijing); Fragment is cloned the expression vector in pEASY-E1 respectively; Through sequencing analysis, its sequence is identical with the corresponding sequence (GenBank:U34036.1) that GenBank announces.Through escherichia coli BL21(DE3) expression, purifying obtain two kinds conjugated protein, said single strand binding protein is made up of 103 amino acid, sees sequence table SEQ ID NO:1, double-stranded conjugated proteinly is made up of 101 amino acid, sees sequence table SEQ ID NO:2.
The preparation of embodiment 2 warm start archaeal dna polymerases (TransStart Taq DNA Polymerase)
Taq DNA Polymerase (day root biochemical technology ltd) is conjugated protein with single-stranded DNA binding protein, the double-stranded DNA of embodiment 1 preparation, according to both having got warm start archaeal dna polymerase ,-20 ℃ of preservations behind the ratio mixing of mol ratio 10-15: 1-2: 1-2.
Embodiment 3
1: enzyme activity determination 1 unit (U) warm start dna polymerase activity is equivalent at 74 ℃, in the 30min with the Oncorhynchi sperm DNA of sensitization as templa-primer, the 10nmol deoxynucleotide is participated in the required enzyme amount of acid non-soluble substance matter.
2: quality control SDS-PAGE detects purity greater than 99%, through detecting no exogenous nucleic acid enzymic activity; PCR method detects no host's residual DNA, the single copy gene in the people's gene group that can increase effectively.
Embodiment 4
The gene of warm start archaeal dna polymerase that embodiment 2 makes (TransStart warm start principle) and Platinum Taq enzyme (antibody sealing method warm start principle) four different lengthss of amplification, relatively both expanding effects.
1: the preparation of human gene group DNA's template and primer
With EasyPure Genomic DNA Kit, extract the genomic dna of Hela cell, and quantitative with ultraviolet spectrophotometer, it is diluted to 50ng/ μ l.
Design primer according to gene order:
Size, the primer of four genes are seen table 1.
2：PCR
Warm start archaeal dna polymerase (embodiment 2 preparations): the PCR reaction system is 50 μ l.
Annotate: 10 * TransStart TopTaq Buffer (500mM Tris-HCl PH 9.0,200Mm (NH)
4SO
4, 20mM MgSO
4, 10% Glycerol)
Platinum Taq DNA enzyme: the PCR reaction system is 50 μ l.
Two kinds of PCR enzymes adopt identical reaction conditionss, and the PCR condition is: hatched 30 minutes 94 ℃ of preparatory sex change 10 minutes for 50 ℃; 94 ℃ 30 seconds, 55 ℃ 30 seconds, 1kb/min is in 72 ℃ of extensions, totally 30 circulations; 72 ℃ 20 minutes; After PCR finishes, respectively get 5 μ l in 1.5% agarose gel electrophoresis detection.
Table 1 pcr amplification the primer, dna fragmentation source and size
Can know that referring to Fig. 2 for same gene, warm start archaeal dna polymerase of the present invention is compared with the PlatinumTaq enzyme, specific amplification is better, and amplification efficiency is higher.
Obviously, the above embodiment of the present invention only be for clearly the present invention is described and is done for example, and be not to be qualification to embodiment of the present invention.For the those of ordinary skill in affiliated field, on the basis of above-mentioned explanation, can also make other multi-form variation or change.Here can't give exhaustive to all embodiments.Everyly belong to the row that conspicuous variation that technical scheme of the present invention extends out or change still are in protection scope of the present invention.
Claims (5)
1. a warm start archaeal dna polymerase is characterized in that, this enzyme comprises:
Can with primer bonded single-stranded DNA binding protein;
Can be conjugated protein with dna profiling bonded double-stranded DNA; And
Archaeal dna polymerase.
2. warm start archaeal dna polymerase according to claim 1 is characterized in that, in said warm start archaeal dna polymerase, said single-stranded DNA binding protein, double-stranded DNA conjugated protein with mol ratio archaeal dna polymerase be 1-2: 1-2: 10-15.
3. warm start archaeal dna polymerase according to claim 1 and 2 is characterized in that: the aminoacid sequence of said single-stranded DNA binding protein is shown in SEQ ID NO:1, and the protein-bonded aminoacid sequence of said double-stranded DNA is shown in SEQ ID NO:2.
4. warm start archaeal dna polymerase according to claim 3 is characterized in that, said archaeal dna polymerase is the Taq archaeal dna polymerase.
5. the application of the described warm start archaeal dna polymerase of the arbitrary claim of claim 1～4 in the heat start PCR reaction.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102876664A (en) * | 2012-10-16 | 2013-01-16 | 北京市农林科学院 | Leaf direct PCR (polymerase chain reaction) method and application of method to transgene plant detection |
| CN103820415A (en) * | 2014-02-25 | 2014-05-28 | 沈鹤霄 | Preparation method of hot start DNA polymerase, prepared polymerase and application |
| CN106350493A (en) * | 2016-08-26 | 2017-01-25 | 周辉 | Hot start Taq DNA polymerase preparation method |
| CN111662386A (en) * | 2020-06-15 | 2020-09-15 | 北京全式金生物技术有限公司 | Taq DNA polymerase monoclonal antibody combination, polymerase reaction system containing same and application thereof |
-
2012
- 2012-06-07 CN CN2012101858148A patent/CN102703400A/en active Pending
Non-Patent Citations (8)
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| 《biochemistry》 20041231 Petrov 等 Diversity of structure and function of DNA polymerase (gp43) of T4-related bacteriophages 1489-1496 第69卷, 第11期 * |
| 《cell》 20010601 Franklin 等 Structure of the replicationg complex of a pol alpha family DNA polymerase 657-667 第105卷, * |
| ABRAMSON AND MYERS: "Nucleic acid amplification technologies", 《BIOTECHNOLOGY》 * |
| FRANKLIN 等: "Structure of the replicationg complex of a pol α family DNA polymerase", 《CELL》 * |
| PETROV 等: "Diversity of structure and function of DNA polymerase (gp43) of T4-related bacteriophages", 《BIOCHEMISTRY》 * |
| SUN 等: "biochemical characterization of interactions between DNA polymerase and single-stranded DNA-binding protein in bacteriophage RB69", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 * |
| TRANSGEN BIOTECH CO.,LTD.: "TransStart Taq DNA polymerase", 《HTTP://TRANSGEN.EN.ALIBABA.COM/PRODUCT/753489330-218181461/TRANSSTART_TAQ_DNA_POLYMERASE.HTML》 * |
| XINYINGSMILE: "cDNA获取方法", 《HTTP://WENKU.BAIDU.COM/VIEW/D6390D4D852458FB770B56F4》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102876664A (en) * | 2012-10-16 | 2013-01-16 | 北京市农林科学院 | Leaf direct PCR (polymerase chain reaction) method and application of method to transgene plant detection |
| CN103820415A (en) * | 2014-02-25 | 2014-05-28 | 沈鹤霄 | Preparation method of hot start DNA polymerase, prepared polymerase and application |
| CN103820415B (en) * | 2014-02-25 | 2017-01-11 | 武汉金开瑞生物工程有限公司 | Preparation method of hot start DNA polymerase, prepared polymerase and application |
| CN106350493A (en) * | 2016-08-26 | 2017-01-25 | 周辉 | Hot start Taq DNA polymerase preparation method |
| CN111662386A (en) * | 2020-06-15 | 2020-09-15 | 北京全式金生物技术有限公司 | Taq DNA polymerase monoclonal antibody combination, polymerase reaction system containing same and application thereof |
| CN111662386B (en) * | 2020-06-15 | 2021-03-30 | 北京全式金生物技术有限公司 | Taq DNA polymerase monoclonal antibody combination, polymerase reaction system containing same and application thereof |
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Application publication date: 20121003 |