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DNA polymerase mutant with improved thermal stability, and construction method and application thereof

A thermal stability and mutant technology, applied in the field of DNA polymerase mutants and their construction, can solve the problems of poor thermal stability of DNA polymerase, etc., and achieve the effects of short culture period, good application prospects and simple culture conditions

Active Publication Date: 2020-04-28
SUZHOU INST OF BIOMEDICAL ENG & TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the defects of poor thermostability of existing DNA polymerases, thereby providing a DNA polymerase mutant with improved thermostability, a method for constructing a DNA polymerase mutant, and a DNA polymerase The use of mutants in single-molecule sequencing

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  • DNA polymerase mutant with improved thermal stability, and construction method and application thereof
  • DNA polymerase mutant with improved thermal stability, and construction method and application thereof

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Embodiment 1

[0039] This embodiment provides a DNA polymerase mutant with improved thermostability, which is a molecularly engineered derivative protein based on the amino acid sequence of the wild-type DNA polymerase (AAA32368.1), wherein the wild Type DNA polymerase (AAA32368.1) is a protein expressed by the wild-type DNA polymerase (AAA32368.1) gene derived from Bacillus phage M2 after codon optimization, and the nucleic acid sequence encoding the protein It is SEQ ID NO.1, and the amino acid sequence is SEQ ID NO.2.

[0040] The DNA polymerase mutants with improved thermostability provided in this example include:

[0041] (a1) a derivative protein having the same function as the protein shown in SEQ ID NO.2 by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO.2; or

[0042] (a2) A derivative protein that substitutes one or more amino acid residues at one or more positions of the amino acid sequence shown in SEQ ID NO.2 and exhibits...

Embodiment 2

[0060] The present embodiment provides a gene encoding the DNA polymerase mutant as described in Example 1:

[0061] (1) The nucleic acid sequence encoding the DNA polymerase single-point mutant whose mutation site is A481K is SEQ ID NO.3;

[0062] (2) The nucleic acid sequence encoding the DNA polymerase single-point mutant whose mutation site is I170L is SEQ ID NO.4;

[0063] (3) The nucleic acid sequence encoding the DNA polymerase single-point mutant whose mutation site is V318A is SEQ ID NO.5;

[0064] (4) The nucleic acid sequence encoding the DNA polymerase single-point mutant whose mutation site is R284F is SEQ ID NO.6;

[0065] (5) The nucleic acid sequence encoding the DNA polymerase single-point mutant whose mutation site is M99E is SEQ ID NO.7;

[0066] (6) The nucleic acid sequence encoding the DNA polymerase single-point mutant whose mutation site is Q499E is SEQ ID NO.8;

[0067] (7) The nucleic acid sequence encoding the DNA polymerase single-point mutant wh...

Embodiment 3

[0076] This embodiment provides a recombinant plasmid comprising the gene provided in Example 2, which is a plasmid vector that can be used to integrate DNA polymerase single point mutants and combined mutant genes into host cells for stable expression, such as pET28a, pET20b, pET9a, pUC18, pUC19, pET15b, pET22a, etc., can also be used to integrate DNA polymerase genes into host cells for expression; host cells can be selected from Gram-negative bacteria (such as E. coli), Gram-positive bacteria (such as Bacillus subtilis), fungi (such as Aspergillus), yeast (such as Saccharomyces cerevisiae), actinomycetes (such as Streptomyces), etc., can be used to express DNA polymerase single point mutants and combined mutant proteins.

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Abstract

The invention belongs to the technical field of biology, and particularly relates to a DNA polymerase mutant with improved thermal stability, and a construction method and application thereof. The DNApolymerase mutant provided by the invention comprises a single-point mutant and a combined mutant, wherein the single-point mutant and the combined mutant have longer half-life at 65 DEG C compared with wild-type DNA polymerase (AAA32368.1); and in particular, the combined mutant shows a superposition effect of thermal stability of the single-point mutant, and the half-life of the combined mutantis about three times that of the wild-type DNA polymerase (AAA32368.1). Therefore, the DNA polymerase mutant provided by the invention has better thermal stability and is suitable for being applied to single molecule nuclear sequence at high temperature.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a DNA polymerase mutant with improved thermostability, a construction method and application thereof. Background technique [0002] High-throughput sequencing technology (High-throughput sequencing), also known as "next-generation" sequencing technology ("Next-generation" sequencing technology), can perform sequence determination and general reading on hundreds of thousands to several million DNA molecules in parallel at a time. Longer and shorter are symbols. Single-molecule sequencing technology (Single-molecule sequencing) is the mainstream development direction of high-throughput sequencing technology. extensive attention and research. The principle of single-molecule sequencing technology is divided into: sequencing-by-synthesis method and direct-reading method. Among them, sequencing-by-synthesis method has the best technical maturity and the highest market accept...

Claims

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Application Information

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IPC IPC(8): C12N9/12C12N15/54C12Q1/6869
CPCC12N9/1252C12Q1/6869C12Y207/07007
Inventor 马富强周连群郭振张艺凡杨广宇唐玉国
Owner SUZHOU INST OF BIOMEDICAL ENG & TECH CHINESE ACADEMY OF SCI
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