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A mutant type Pfu DNA polymerase and a preparing method and application thereof

A mutant and polymerase technology, applied in the biological field, can solve the problems of inability to amplify long fragments of DNA, poor resistance to inhibitors, slow extension rate, etc.

Active Publication Date: 2018-12-18
广州英赞生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, wild-type Pfu DNA polymerase also has many defects when performing PCR reactions, such as slow elongation rate, insufficient elongation ability, inability to amplify long fragments of DNA, and poor resistance to inhibitors. Low amplification efficiency in the PCR reaction

Method used

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  • A mutant type Pfu DNA polymerase and a preparing method and application thereof
  • A mutant type Pfu DNA polymerase and a preparing method and application thereof
  • A mutant type Pfu DNA polymerase and a preparing method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Embodiment 1 constructs the recombinant vector containing the nucleotide sequence encoding mutant Pfu DNA polymerase of the present invention

[0069] (1) According to the amino acid sequence of the mutant Pfu DNA polymerase of the present invention, the codon optimization of the expression system of E. coli is carried out to obtain a DNA molecule capable of high-efficiency expression in E. coli, and the method of splicing PCR is used to artificially synthesize the encoded mutant The DNA molecule of Pfu DNA polymerase is specifically shown in SEQ ID NO:2.

[0070] (2) Recombining the DNA molecule encoding the mutant Pfu DNA polymerase of the present invention with the expression vector pET-28a. The synthetic primer sequences are as follows:

[0071] Nde I-FP: 5'-GCGCCATATGATTTTAGATGTGGATTAC-3';

[0072] Sal I-RP: 5'-GCGCGTCGACTCATTTTTTCTGCTT-3'.

[0073] The DNA molecule of the mutant Pfu DNA polymerase of the present invention is amplified by PCR, and the artificial...

Embodiment 2

[0075] Embodiment 2 Expression and identification of mutant Pfu DNA polymerase of the present invention in recombinant Escherichia coli

[0076] (1) Inoculate the positive transformant strain capable of expressing the mutant Pfu DNA polymerase of the present invention obtained in Example 1 into 60 mL LB medium containing 50 μg / mL kanamycin, and place it in a shaker at 37° C. Cultivate overnight to obtain seed solution.

[0077] (2) Take the overnight cultured seed solution and inoculate it into 500 mL LB medium containing 50 μg / mL kanamycin at a volume ratio of 1:100, and continue shaking culture in a shaker at 37 ° C for 4 h, OD 600 is 0.8.

[0078] (3) Add IPTG to each bottle of bacterial solution to a final concentration of 0.1 mmol / L, and continue shaking induction at 18°C ​​for 18 hours.

[0079] (4) Centrifuge, collect and weigh the induced bacteria, and record the wet weight of the bacteria. Take 1 mL of each bacterial solution before and after induction, centrifuge at...

Embodiment 3

[0081] Embodiment 3 Mutant type Pfu DNA polymerase purification of the present invention

[0082] (1) Ultrasonic disruption of recombinant bacteria

[0083] Take the induced expression cells frozen at -20°C, according to the wet weight of the cells recorded in Example 2, add 5 mL of lysis buffer to resuspend the cells per gram of the cells, and lyse the cells with an ultrasonic cell disruptor. The ultrasonic conditions are: : Power 250W, ultrasound 5s, interval 5s, last 30min. Put the lysed bacteria into a high-speed refrigerated centrifuge, centrifuge at 12000r / min for 30min at 4°C, and take the supernatant into a 250mL sterilized flask. The supernatant was incubated in a constant temperature water bath at 75°C for 30min, centrifuged at 12000r / min for 30min at 4°C, filtered through a 0.22μm microporous membrane, and the supernatant was taken into a 250mL sterile solvent bottle.

[0084] (2) Nickel ion affinity chromatography purification

[0085] Choose HisTrap TM HP 5mL...

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Abstract

A mutant type Pfu DNA polymerase and a preparing method and application thereof are disclosed. The amino acid sequence of the mutant type Pfu DNA polymerase is shown as SEQ ID NO:1. The mutant type Pfu DNA polymerase has greatly improved DNA polymerase amplification speed, extending capability and fidelity, and is suitable for various types of conventional PCR, high-fidelity PCR, long-fragment PCRand PCR with a high-GC template. In addition, the mutant type Pfu DNA polymerase has high tolerance to PCR inhibitors in blood, and a blood sample can be directly utilized for PCR detection without the need of DNA purification in advance, thus saving time, and avoiding cross contamination during nucleic acid extraction. The mutant type Pfu DNA polymerase is suitable for direct DNA amplification in anticoagulated blood or blood collected by conventional filter paper, and is suitable for genetic disease diagnosis and paternity tests.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a mutant Pfu DNA polymerase and its preparation method and application. Background technique [0002] Polymerase chain reaction (PCR) can easily, quickly and accurately replicate target gene fragments on a large scale, and has become an indispensable experimental technique in modern molecular biology. Widely used in many fields. [0003] When PCR technology is applied to the amplification of blood-derived genes, since blood samples contain substances that can inhibit PCR reactions such as hemoglobin, immunoglobulin G, lactoferrin, and anticoagulants, the genome in the sample needs to be purified before PCR reaction. DNA. However, this kind of purification often cannot completely remove the adverse effects of inhibitors on PCR amplification, and the purification is also likely to cause cross-contamination of samples, which affects the accuracy and reliability of PCR results. [0004]...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12N15/54C12N15/70C12N1/21C12N15/10C12Q1/686C12R1/19
CPCC12N9/1252C12Q1/686C12Y207/07007C12Q2521/101
Inventor 胡松青黄丽芳刘光毅黄菁菁侯轶
Owner 广州英赞生物科技有限公司
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