Mutant Taq DNA (deoxyribonucleic acid) polymerase, method for preparing same and application of mutant Taq DNA polymerase

A mutant and polymerase technology, applied in the biological field, can solve problems such as limitations, low activity, and low tolerance

Active Publication Date: 2015-11-11
FAPON BIOTECH INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Structural defects of wild-type TaqDNA polymerase limit its application in many fields
The existing TaqDNA polymerase has low activity and low tolerance, which cannot meet the needs of industrial production and scientific research applications

Method used

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  • Mutant Taq DNA (deoxyribonucleic acid) polymerase, method for preparing same and application of mutant Taq DNA polymerase
  • Mutant Taq DNA (deoxyribonucleic acid) polymerase, method for preparing same and application of mutant Taq DNA polymerase
  • Mutant Taq DNA (deoxyribonucleic acid) polymerase, method for preparing same and application of mutant Taq DNA polymerase

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preparation example Construction

[0059] In addition, the present invention also provides figure 1 The preparation method of above-mentioned mutant type TaqDNA polymerase shown, comprises the steps:

[0060] S110, providing a gene expression sequence for expressing the mutant TaqDNA polymerase.

[0061] Generally, the gene expression sequence can select the corresponding gene sequence from the gene bank (GeneBank) according to the mutant TaqDNA polymerase that needs to be expressed, and design mutation primers as needed to point-mutate one or a certain base sequence.

[0062] In one embodiment, the gene expression sequence comprises:

[0063] (a), the nucleotide sequence shown in SEQIDNo.1;

[0064] (b), a nucleotide sequence having at least 98% homology to the nucleotide sequence shown in SEQ ID No.1; or

[0065] (c) The nucleotide sequence shown in SEQIDNo.1, wherein one or more bases are deleted, substituted or added.

[0066] In one embodiment, mutant TaqDNA polymerase comprises:

[0067] (a), a polyp...

Embodiment 1

[0106] The preparation of embodiment 1 mutant type TaqDNA polymerase gene expression vector

[0107] The plasmid template, mutation primers and / or splicing sequence of the wild-type TaqDNA polymerase gene (GenBank: D32013.1) were selected. Prepare the PCR reaction system, the PCR reaction system is 50μL, including 10×buffer (100mMKCl, 100mM(NH4) 2 SO 4 , 200mM Tris-HClpH8.8, 20mM MgSO 4 , 1% TritonX-100, 1mg / mL BSA) 5μL, 10mMdNTP0.5μL, mutation primers (splicing primers) each 125ng, 1Upfu enzyme, containing 50ng plasmid template 1μL, add ddH 2 0 to 50 μL. Mutation primers and splicing sequences are artificially synthesized oligonucleotides, which contain the sequence of the site to be mutated and the site to be inserted, and the bases to be mutated have been replaced. The conditions of PCR amplification were denaturation at 95°C for 30s, and the cycle cycle was 95°C, 30s, 55°C, 1min, 68°C, 14min, 25 cycles. After the PCR reaction, place the reaction tube on ice to keep th...

Embodiment 2

[0108] Expression and purification of embodiment 2 mutant TaqDNA polymerase

[0109] The mutant TaqDNA polymerase gene expression vector was transformed into Escherichia coli competent cell BL21 (Clauning (Beijing) Biotechnology Co., Ltd., catalog number BL21-100). After resistance screening, take 50 μL strains and inoculate them in 5 mL LB liquid medium for 6-8 hours, then transfer them to 250 mL LB liquid medium for 4-6 hours, then add IPTG to a final concentration of 50 mM and continue shaking overnight . Centrifuge 250mL of bacterial liquid in a 50mL centrifuge tube at 5000rpm for 10min to collect the bacteria, add 5mL of Bindingbuffer per 100mL of culture, add lysozyme to a final concentration of 1mg / mL, place on ice for 1h, shake at 4°C for 10min, add TritonX-100 to the final concentration Shake and mix vigorously, oscillate at 4°C for 10 minutes, centrifuge at 5000rpm for 30min, take the supernatant (reserved sample of 20μL bacterial cell crushing product), bathe in 75...

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Abstract

The invention discloses a mutant Taq DNA (deoxyribonucleic acid) polymerase. Amino acid sequences of a wild Taq DNA polymerase are mutated, particularly, 605th leucine of the wild Taq DNA polymerase is mutated to obtain arginine, 617th arginine of the wild Taq DNA polymerase is mutated to obtain phenylalanine, 667th phenylalanine of the wild Taq DNA polymerase is mutated to obtain leucine, and 480th-485th amino acid of thumb zones of the Taq DNA polymerase is replaced by TBD zones, namely, 262nd-337th amino acid of T3 DNA polymerase. The mutant Taq DNA polymerase has the advantages that the activity, the tolerance and the like of the mutant Taq DNA polymerase can be obviously improved owing to site-directed mutation; the mutant Taq DNA polymerase with the high enzyme activity and the high tolerance can be used in direct amplification PCR (polymerase chain reaction) systems without nucleic acid extraction. The invention further discloses a method for preparing the mutant Taq DNA polymerase and application thereof.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a mutant TaqDNA polymerase and its preparation method and application. Background technique [0002] Taq DNA Polymerase is a heat-resistant DNA polymerase isolated from Thermus aquaticus. In 1969, a Thermus aquaticus YT-1 was isolated from the volcanic hot springs of the Yellowstone National Forest Park in the United States. It can grow in an environment rich in minerals at 70°C to 75°C. In 1976, A. Chien isolated and purified thermostable TaqDNA polymerase from Thermus aquaticus. In 1983, karyMullis invented the polymerase chain reaction (PCR). In 1988, Randall K. Saiki et al. also isolated and purified TaqDNA polymerase from Thermus aquaticus and applied it to PCR technology, realizing the automatic continuous cycle of the PCR process. Existing studies have shown that Taq DNA polymerase belongs to the DNA polymerase I family, the enzyme gene has a full length of 2496 bases, encod...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12N15/54C12N15/70C12N15/10
CPCC12N9/1252C12Y207/07007
Inventor 邓艳华杨浩李泓彦
Owner FAPON BIOTECH INC
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