Mutant taq DNA polymerase, nucleic acid extraction-free direct PCR amplification kit and application thereof

A polymerase and mutant technology, applied in the biological field, can solve the problems of increased cost of the kit, low tolerance, difficult preparation and the like

Active Publication Date: 2020-07-14
深圳市艾伟迪生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with the traditional method, although the operation is much simpler, the cost of the kit has also increased a lot due to the cost of source transportation and the distance of transportation time.
In addition, DNA polymerases with high tolerance generally require more mutation sites and are not easy to prepare, which brings inconvenience to many of the above-mentioned research and detection fields.
[0004] In summary, traditional DNA polymerases have low activity, low tolerance, and are not easy to prepare

Method used

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  • Mutant taq DNA polymerase, nucleic acid extraction-free direct PCR amplification kit and application thereof
  • Mutant taq DNA polymerase, nucleic acid extraction-free direct PCR amplification kit and application thereof
  • Mutant taq DNA polymerase, nucleic acid extraction-free direct PCR amplification kit and application thereof

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preparation example Construction

[0041] One embodiment of the method for preparing mutant Taq DNA polymerase includes the following steps S110-S120.

[0042] S110. Using the gene expression sequence of Taq DNA polymerase as the amplification template, and adding point mutation primers to perform point mutation PCR amplification to obtain the target gene expression sequence; wherein, the protein encoded by the target gene expression sequence is the same as the wild-type Taq DNA The glutamic acid at position 709 was mutated to glutamine compared to the polymerase.

[0043] S120, transforming the target gene expression sequence obtained in S110 into a host cell, and inducing expression of the transformed host cell to obtain a mutant Taq DNA polymerase.

[0044] Specifically, search the gene sequence of Taq DNA Polymerase on NCBI, and perform codon optimization to design the gene coding sequence of mutant Taq DNA polymerase. Use the glutamic acid E mutation at position 709 of Taq DNA Polymerase to design two poi...

Embodiment 1

[0066] The preparation of embodiment 1 mutant type Taq DNA polymerase

[0067] Find the gene sequence of Taq DNA Polymerase on NCBI (GenBank sequence number: D32013.1), and optimize the codons to design the target gene expression sequence. Use the glutamic acid E mutation at position 709 of Taq DNA Polymerase to design two point mutation primers for glutamine Q as follows:

[0068] F-primer: CGAAAGTTCGCGCTTGGATC CAA AAAACCCTGGAAGAAGG (as shown in SEQ ID No.3)

[0069] R-primer: CCTTCTTCCAGG GTT TTTTGGATCCAAGCGCGAACTTTCG (as shown in SEQ ID No.4)

[0070] Synthesized by Shanghai Sangon Bioengineering Co., Ltd. Using the PTaq DNA Polymerase gene extracted by our company as a template, the above-mentioned pair of complementary primers are used for point mutation PCR amplification to obtain the point mutation product RiTaq (target gene expression sequence), and the PTaq template is removed with the restriction endonuclease Dnp I , after purifying the product, sequence it, r...

Embodiment 2

[0072] Detection Kit for Direct PCR Amplification Without Nucleic Acid Extraction

[0073] The kit includes mutant Taq DNA polymerase, treatment solution, PCR enhancer and dNTP in Example 1, and the treatment solution contains 50mmol / L Tris-HCl (pH 9.1), 25mmol / L MgCl 2 , 160mmol / L (NH4 ) 2 SO 4 and 0.25% Brij-58. The PCR enhancer is betaine.

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Abstract

The invention discloses mutant type Taq DNA polymerase, a nucleic acid extraction-free direct PCR amplification kit and an application of the mutant type Taq DNA polymerase. By mutating 709-site glutamic acid of wild Taq DNA polymerase into glutamine, the activity, tolerance and the like of the obtained mutant type Taq DNA polymerase are obviously improved. The mutant type Taq DNA polymerase onlyhas a site, so that the mutation sites are few, and the preparation is easy. Experiment results show that the mutant type Taq DNA polymerase can be applied to a nucleic acid extraction-free direct PCRamplification reaction system, and the nucleic acid extraction-free direct PCR amplification of samples such as blood, foods and soil which contain a large number of inhibiting factors can be realized.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a mutant Taq DNA polymerase, a nucleic acid extraction-free direct PCR amplification kit and applications thereof. Background technique [0002] Polymerase chain reaction (PCR) is one of the most important tools in the field of molecular biology research. In the PCR reaction, the activity characteristics of DNA polymerase are related to the specificity, efficiency and fidelity of the PCR reaction. Taq DNA polymerase is a highly thermostable DNA polymerase derived from the thermophilic bacterium Thermus aquaticus, and is widely used in PCR amplification due to its high temperature resistance, high specificity and sensitivity. However, conventional Taq DNA polymerase is limited by inhibitors in the amplification template, such as humic acid in soil, various components in mouse serum, animal and plant tissues, etc. Contains a large number of inhibitory factors, such as hemoglobin, meth...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/12C12N15/54C12N15/63C12Q1/686
CPCC12N9/1252C12Q1/686C12Y207/07007C12Q2527/125C12Q2521/101
Inventor 谭毅彬马柳安郑林李泓彦周娇娇
Owner 深圳市艾伟迪生物科技有限公司
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