Method for preparing hot start Taq polymerase

A hot start and heparin technology, applied in biochemical equipment and methods, enzymes, transferases, etc., to improve reaction specificity, reduce human error, and improve amplification efficiency

Active Publication Date: 2013-04-17
成都峰际生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] The methods commonly used above can only be hot-started in the first step of PCR reaction, and it is still difficult to avoid the occurrence of non-specific sequence amplification in the subsequent steps

Method used

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  • Method for preparing hot start Taq polymerase

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Effect test

Embodiment 1

[0025] The invention relates to a method for preparing a hot-start Taq enzyme. The Taq enzyme is mixed with a treatment solution to obtain the hot-start Taq enzyme.

[0026] The treatment solution is heparin solution, heparin sodium solution or heparin potassium solution, and the concentration of heparin, heparin sodium or heparin potassium in the mixture of Taq enzyme and treatment solution is above 0.0001 U / ml.

[0027] Its specific operation method is:

[0028] Heparin, heparin sodium or heparin potassium is dissolved in deionized water until the final concentration of the solution is above 0.0001U / ml, and Taq enzyme is added into the solution. The reaction product is purified by dialysis, column or ultrafiltration to obtain the hot start Taq enzyme.

Embodiment 2

[0030] The invention relates to a method for preparing a hot-start Taq enzyme. The Taq enzyme is mixed with a treatment solution to obtain the hot-start Taq enzyme.

[0031] The treatment solution is heparin solution, heparin sodium solution or heparin potassium solution, and the concentration of heparin, heparin sodium or heparin potassium in the mixture of Taq enzyme and treatment solution is above 0.0001 U / ml.

[0032] Its specific operation method is:

[0033] Add heparin, heparin sodium or heparin potassium to the PCR reaction solution, so that the final concentration of heparin, heparin sodium or heparin potassium is above 0.0001U / ml, after the Taq enzyme in the PCR reaction solution is mixed with heparin, heparin sodium or heparin potassium solution , that is, hot-start Taq enzyme.

Embodiment 3

[0035] Apply the hot-start Taq enzyme of the present invention to PCR, take Foregene PCR Easy as an example, use template 2.5ul (2.5ng), 2×PCR Easy Mix 25ul, 10uM Primer 1 1ul, 10uM Primer 2 1ul, add ddH 2 From 0 to 50ul, react according to the following reaction conditions: thermal denaturation at 94°C for 3min, denaturation at 94°C for 30sec, annealing at 55°C for 30sec, extension at 72°C for 1min, a total of 25-40 cycles, and finally hold at 72°C for 5min. At the same time, untreated DNA polymerase (5U / ul) was used as a parallel control. After completing the PCR reaction, take 5ul of the reaction product and test it by agar gel electrophoresis. The results are shown in figure 1 ,from figure 1 It can be seen that the amplification intensity of the hot-start high-temperature-resistant DNA polymerase is significantly increased. In the figure, 1, 2, 3: ordinary Taq enzyme amplification, 1a, 2a, 3a: hot start Taq enzyme amplification, M: 100bp-1KB Marker, 1-1a: ZmIVR (225bp), ...

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Abstract

The invention discloses a method for preparing a hot start Taq polymerase. The method comprises the following step of mixing the Taq polymerase with a a treatment solution, so as to obtain the hot start Taq polymerase, wherein the treatment solution is a heparin solution, a heparin sodium solution or a heparin potassium solution. The method for preparing the hot start Taq polymerase has the beneficial effects that the hot start can be realized in the first step of PCR (Polymerase Chain Reaction) reaction during carrying out the PCR reaction, and the hot start also can be realized in the follow-up steps of the PCR reaction, so that the amplification of non-specific sequences can be effectively avoided, and the reaction specificity, reliability, uniformity and sensitivity of the DNA (Deoxyribose Nucleic Acid) polymerase can be improved; compared with the conventional similar product, the hot start Taq polymerase prepared by the method disclosed by the invention has the advantages that the amplification efficiency is increased, the hot start Taq polymerase can be detected from a micro sample and trace sample under relatively low cycle number as well as be cloned to a target gene, thus the personal error can be improved to a great extent; and the hot start Taq polymerase is suitable for being applied to amplification and detection of general PCR reaction, complex templates and trace templates.

Description

technical field [0001] The invention relates to the technical field of preparation methods of Taq enzymes, in particular to a method for preparing hot-start Taq enzymes. Background technique [0002] Polymerase chain reaction, Polymerase chain reaction, referred to as PCR, is a molecular biology technique used to amplify specific DNA fragments. This method can be carried out in vitro without relying on organisms such as Escherichia coli or yeast. The technology of PCR is widely used in medical and biological laboratories, such as for determining whether a certain genetic disease will appear in a sample, diagnosis of infectious diseases, gene replication, and paternity testing. [0003] PCR is used to amplify a small known segment of DNA, which may be a single gene, or just a part of a gene. Unlike living organisms, PCR can only replicate very short DNA fragments, usually no more than 10kbp. The currently applied PCR reaction requires several basic components: DNA template ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12
CPCC12N9/1252
Inventor 邹利平童玉成
Owner 成都峰际生物技术有限公司
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