Tolerance-improved mutant Taq DNA polymerase as well as preparation method and application thereof

A polymerase and mutant technology, applied in the biological field, can solve the problem of low tolerance of TaqDNA polymerase, achieve high polymerization amplification ability, low false negative rate, and improve the effect of impurity tolerance

Active Publication Date: 2020-01-14
VAZYME BIOTECH NANJING
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

SYBR Green fluorescent dye is a commonly used dye for qPCR, but Taq DNA polymerase has little tolerance to it

Method used

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  • Tolerance-improved mutant Taq DNA polymerase as well as preparation method and application thereof
  • Tolerance-improved mutant Taq DNA polymerase as well as preparation method and application thereof
  • Tolerance-improved mutant Taq DNA polymerase as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Construction of a vector containing a nucleotide sequence encoding mutant Taq DNA polymerase

[0046] Using the method of gene synthesis, the synthetic primer sequence is as follows:

[0047] R266F-1: CGGGAGCCCGACTTCGAGAGGCTTAGGGCCTTTCTG (SEQ ID NO. 5);

[0048] R266F-2: CCTAAGCCTCTCGAAGTCGGGCTCCCGCCTTTTGGC (SEQ ID NO. 6);

[0049] A293N-1: GAAAGCCCCAAGAACCTGGAGGAGGCCCCCTGGCCC (SEQ ID NO. 7);

[0050] A293N-2: GGCCTCCTCCAGGTTCTTGGGGCTTTCCAGAAGGCC (SEQ ID NO. 8);

[0051] A414F-1: GAGAGGCTCTTTCTTCAACCTGTGGGGGAGGCTTGAG (SEQ ID NO. 9);

[0052] A414F-2: CCCCCACAGGTTGAAGAAGAGCCTCTCGGAAAGGGC (SEQ ID NO. 10);

[0053] E466Q-1: GAGGTGGCCGAGCAGATCGCCCGCCTCGAGGCCGAG (SEQ ID NO. 11);

[0054] E466Q-2: GAGGCGGGCGATCTGCTCGGCCACCTCCAGGGACAA (SEQ ID NO. 12);

[0055] E507K-1: ATCGGCAAGACGAAGAAGACCGGCAAGCGCTCCACC (SEQ ID NO. 13);

[0056] E507K-2: CTTGCCGGTCTTCTTCGTCTTGCCGATGGCGGGAAG (SEQ ID NO. 14);

[0057] D732E-1: CGCTACGTGCCAGAGCTAGAGGCCCGGGTGAAGAGC (SEQ ID NO....

Embodiment 2

[0062] Embodiment 2 obtains mutant type Taq DNA polymerase

[0063] Take 10ng of the vector verified by sequencing and transform it into BL21(DE3) competent cells (produced by Nanjing Nuoweizan Biotechnology Co., Ltd., Vazyme, catalog number C504) with the same transformation method, and spread it on the antibiotic-resistant cell line containing the corresponding plasmid. Incubate overnight at 37°C on LB plates. Single clones were picked the next day. After being cultured in a liquid medium and induced to express, the purified enzyme of the mutant Taq DNA polymerase is obtained. The mutant Taq DNA polymerase is named Taq-Mut, which consists of 832 amino acids, and its specific sequence is shown in SEQ ID NO.3.

Embodiment 3

[0064] Embodiment 3 blood tolerance performance

[0065] Taq-Mut was tested for its ability to amplify a 320bp PCR amplicon in a PCR reaction using freshly collected human whole blood. Reactions were performed in a buffer containing 20 mM Tris-HCl (pH 8.4) and 20 mM KCl. Exemplary reaction components are shown in Table 1, where Taq DNA polymerase was added in amounts of 60, 40 and 20 ng, respectively. An exemplary cycling procedure for this assay is shown in Table 2.

[0066] Exemplary primer sequences are as follows:

[0067] Primer-L: TAGTGGTGGCTGACCTGTTCTCT (SEQ ID NO. 17)

[0068] Primer-R: TCGTCGATCTCCTGTTGGACA (SEQ ID NO. 18)

[0069] Table 1 Example of PCR detection system. Total volume is 50 μL

[0070]

[0071]

[0072] Table 2 Example of PCR cycling process

[0073]

[0074] Run the reaction product on an agarose gel and assess the presence of the product and the band intensity of the correct fragment size. Exemplary results are shown in figure 1 midd...

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Abstract

The invention discloses tolerance-improved mutant Taq DNA polymerase as well as a preparation method and application thereof. According to the tolerance-improved mutant Taq DNA polymerase, one or moreamino acids are inserted, substituted or deleted in an amino acid sequence of Taq DNA polymerase shown in SEQ ID NO.1, and compared with the Taq DNA polymerase shown in SEQ ID NO.1, the amino acid sequence has significantly enhanced impurity tolerance. The recombinant Taq DNA polymerase mutant has remarkably enhanced tolerance to blood, fluorochrome and high ion strength, and can directly performPCR detection on blood samples, save time and avoid false negative.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a mutant Taq DNA polymerase with improved tolerance, a preparation method and application thereof. Background technique [0002] DNA polymerases are a class of enzymes that use single-stranded DNA as a template to synthesize complementary DNA strands. DNA polymerase can add free nucleotides to the 3' end of the newly formed strand, extending the new strand in the 5'-3' direction. Most DNA polymerases are multifunctional proteins with polymerization and exonuclease activity (eg, 3'-5' exonuclease or 5'-3' exonuclease activity). [0003] Like other natural enzymes, DNA polymerase has evolved over millions of years to function in its natural cellular environment. Many of them are almost perfectly adapted to work in that environment. However, when DNA polymerase is extracted from its natural environment and used in industry or research, the operating environment and conditions of the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12N15/54C12N15/70C12N1/21C12Q1/686C12R1/19
CPCC12N9/1252C12N15/70C12Q1/686C12Y207/07007C12Q2521/101C12Q2563/107
Inventor 贡怡冯速徐晓昱刘来花曹林张力军聂俊伟瞿志鹏
Owner VAZYME BIOTECH NANJING
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