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Anti-serum interference Taq DNA polymerase and preparation and application thereof

A polymerase and antiserum technology, applied in the field of genetic engineering, can solve the problems of low anti-interference ability and insufficient polymerase activity, and achieve the effects of strong anti-interference ability, good thermal stability, and high enzyme expression and purification efficiency.

Active Publication Date: 2018-05-25
WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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  • Description
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Problems solved by technology

However, with the increasing popularization and application of PCR technology, researchers have continuously improved the performance requirements of Taq DNA polymerase, so researchers are constantly looking for new heat-resistant DNA polymerases to make up for some of the defects of wild-type Taq DNA polymerase. Such as polymerase activity is not high enough, low anti-interference ability, etc.

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  • Anti-serum interference Taq DNA polymerase and preparation and application thereof
  • Anti-serum interference Taq DNA polymerase and preparation and application thereof
  • Anti-serum interference Taq DNA polymerase and preparation and application thereof

Examples

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Effect test

Embodiment 1

[0040] Embodiment 1: the preparation of the Taq DNA polymerase gene expression vector of anti-serum interference

[0041] Generally, the gene expression sequence can be selected from the gene bank (GeneBank) according to the mutant Taq DNA polymerase that needs to be expressed, and mutation primers can be designed as needed to mutate one or a certain base sequence.

[0042] In this example, the gene expression sequence was prepared by the following operations:

[0043] 1. Obtain the nucleotide sequence shown in SEQ ID NO: 1, and clone the sequence

[0044] Sampling location: Rehai, Tengchong City, Yunnan Province (1390m above sea level)

[0045] Soil mixed samples: pregnant tire well (88°C), Pearl Spring (98°C), Guming Spring (85°C), Hamozui No. 4 hillside soil (79.9°C), mix well and take 10g for later use.

[0046] Water samples: Hamozui No. 1 (65.2°C), Hamozui No. 3 (88°C), Hamozui No. 4 hillside water (79.9°C), pregnant well left (91.7°C), pregnant well right (78.7°C), Pe...

Embodiment 2

[0054] Example 2: Expression and purification of Taq DNA polymerase interfered with by antiserum

[0055] Refer to the method recommended by the kit manufacturer for the gene expression vector transformation operation. The host cells are competent cells, such as E. The structure of the cell membrane is disturbed, and a gap appears on the cell membrane to allow the gene expression vector to enter the cell, and then cultured at a constant temperature to revive the host cell. The specific method for the expression and purification of Taq DNA polymerase interfered by antiserum in this embodiment is as follows:

[0056] The Taq DNA polymerase gene expression vector (pET28a-Taq DNA) obtained by metagenomic analysis was transformed into Escherichia coli competent cell BL21. For resistance screening, single clones were picked and cultured in 5mL LB liquid medium at 37°C and 180rpm for 8-10h, then transferred to 500mL LB liquid medium, and cultured at 37°C and 180rpm for about 2h. Whe...

Embodiment 3

[0057] Example 3: SDS-PAGE protein electrophoresis to verify the protein size and purity of Taq DNA polymerase against serum interference

[0058] The Taq DNA polymerase obtained above was subjected to SDS-PAGE protein electrophoresis detection to identify the size and purity of the mutant Taq DNA polymerase protein.

[0059] The SDS-PAGE stacking gel was used at a concentration of 5%, and the separation gel was used at a concentration of 8%. Take 40 μL of the dialyzed protein sample and mix with 10 μL of 5× protein loading buffer, boil for 10 min to prepare the sample. After the sample is prepared, take 10 μL, load the sample together with the protein marker (ProteinRuler II (12-120kDa), Thermo Scientific Fermentas), and electrophoresis at a constant voltage of 50V for 30min. 150V constant electrophoresis to the bottom of the separation gel. After the electrophoresis is completed, carefully pull out the gel and stain it with Coomassie Brilliant Blue staining solution on a s...

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Abstract

The invention relates to an anti-serum interference Taq DNA polymerase analyzed and obtained from a hot-spring meta genome, and relates to the anti-serum interference Taq DNA polymerase and a preparation method and application thereof. The protein amino acid sequence of the anti-serum interference Taq DNA polymerase is shown as SEQ ID No:1; the genes of the anti-serum interference Taq DNA polymerase are coded, and the nucleotide sequence is shown as SEQ ID No:2. Compared with the amino acid sequence of a wild Taq DNA polymerase, the protein amino acid sequence of the polymerase has 13 amino acid mutations. The sequence is cloned and subjected to polymerase expression and purification, and the polymerase has higher polymerase activity than a commercial Taq DNA polymerase and the wild Taq DNA polymerase; the polymerase is used for simulated sample detection in serum, and the result of a fluorogenic quantitative PCR shows that the polymerase has better tolerance than the wild Taq DNA polymerase and the commercial polymerase. Therefore, the polymerase can be used for conducting direct PCR detection on a nucleic acid extraction-free serum sample.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to an anti-serum interference Taq DNA polymerase obtained from hot spring metagenome analysis, and to a preparation method and application of the anti-serum interference Taq DNA polymerase. Background technique [0002] Taq DNA polymerase is a heat-resistant DNA polymerase isolated by scientist A.Chien from the extreme thermophile Thermus aquaticus in 1976 (Chien A, Edgar DB, Trela ​​JM. Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus.[J ]. Bacteriol, 1976, 127(3): 1550-1557.). In 1983, Kary Mullis invented PCR (polymerase chain reaction). In 1988, Randall K. Saiki and others applied Taq DNA polymerase to PCR technology for the first time, making the PCR process realize automatic continuous cycle. Taq DNA polymerase is a heat-resistant DNA polymerase belonging to the DNA polymerase I family. The enzyme gene is 2496 bases in length, encodi...

Claims

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Application Information

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IPC IPC(8): C12N9/12C12N15/54C12N15/70
CPCC12N9/1252C12Y207/07007
Inventor 危宏平余军平熊进张晓旭
Owner WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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