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Warm starting TaqDNA polymerase and its preparation method and application

A polymerase and hot-start technology, which is applied in the fields of biochemistry and molecular biology, can solve the problems of interfering with the amplification of target fragments and the inability of specific bands to expand, so as to improve specificity, avoid non-specific amplification products, The effect of increasing production

Active Publication Date: 2018-03-23
东北制药集团辽宁生物医药有限公司
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Problems solved by technology

[0003] Before the denaturation of the first cycle of PCR, there will be some non-specific pairing between primers and templates during the heating process. If Taq DNA polymerase is active at this time, non-specific amplification will easily occur. Since the amount of templates in the initial cycle is very small, the resulting Subsequent exponential amplification of non-specific bands will seriously interfere with the amplification of target fragments, and even cause specific bands to fail to amplify

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  • Warm starting TaqDNA polymerase and its preparation method and application
  • Warm starting TaqDNA polymerase and its preparation method and application
  • Warm starting TaqDNA polymerase and its preparation method and application

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Embodiment 1

[0060] The preparation method of embodiment 1 hot start Taq DNA polymerase

[0061] In this example, the antibody modification method was used to prepare hot-start Taq DNA polymerase. According to the Taq DNA polymerase gene sequence (D32013.1) published by GenBank, the optimized Taq DNA polymerase gene fragment was artificially synthesized according to the preferred codons of Escherichia coli; then the optimized Taq DNA polymerase gene fragment was constructed on a prokaryotic expression vector, Transformation and screening of Escherichia coli were carried out to obtain recombinant engineering bacteria expressing Taq DNA polymerase; followed by inductive expression, cell wall breaking, thermal denaturation, gradient salting out, dialysis and chromatography to complete the concentration and purification of Taq DNA polymerase. Purification; finally, the anti-Taq DNA polymerase monoclonal antibody is specifically combined with the Taq DNA polymerase to complete the ligand modifi...

Embodiment 2

[0082] Example 2 Application of Hot Start Taq DNA Polymerase in DNA Fluorescence Quantitative Detection

[0083] 1. Enzyme activity calibration

[0084] Using the Hot Start Taq enzyme (5U / μl) from Dalian Bao Biological Co., Ltd. as the standard, the enzyme activity of the prepared Taq DNA polymerase finished product was calibrated by using the fluorescent quantitative PCR method. The detection mean value of the Taq DNA polymerase finished product and the detection mean value of the Hot Start Taq enzyme of Dalian Bao Biological Co., Ltd. were log-logarithmically fitted, and the linear correlation coefficient R>0.97 was required.

[0085] 1.1 Sample preparation

[0086] HBV DNA serum standard substance (concentration: 1.2×10 6 IU / ml), carry out 10-fold serial dilution with negative serum, dilute 4 concentrations altogether (1.2×10 5 IU / ml, 1.2×10 4 IU / ml, 1.2×10 3 IU / ml, 1.2×10 2 IU / ml), 1.2×10 5 IU / ml~1.2×10 2 The 4 concentrations of IU / ml were tested three times in ...

Embodiment 3

[0107] Example 3 Application of Hot Start Taq DNA Polymerase in RNA Fluorescence Quantitative Detection

[0108] 1. RNA virus sample preparation

[0109] Hepatitis C virus (HCV) is a single-stranded positive-sense RNA virus. The HCV minimum detection limit reference product of China National Institutes for Food and Drug Control was used as the initial sample (concentration of 6×10 6 IU / ml), carry out 10-fold serial dilution with negative serum, dilute 5 concentrations altogether (6×10 5 IU / ml, 6×10 4 IU / ml, 6×10 3 IU / ml, 6×10 2 IU / ml, 6×10 1 IU / ml).

[0110] 2. HCV RNA extraction

[0111] The "nucleic acid extraction kit (magnetic bead method)" produced by Northeast Pharmaceutical Group Liaoning Biomedical Co., Ltd. was used for the extraction and purification of hepatitis C virus nucleic acid. For specific operations, refer to the product manual.

[0112] 3. Adding samples and testing

[0113] Prepare the HCV RT-PCR reaction solution according to Table 3, then take ...

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Abstract

The invention relates to a warm starting TaqDNA polymerase and its preparation method and an application. The warm starting TaqDNA polymerase is prepared by an antibody modification method, and includes steps of (1), structuring and transformation of a recombination expression carrier; (2), inductive expression of recombinant bacteria; (3), preparation and purification of crude enzyme; (4), antibody modification. The warm starting TaqDNA polymerase is high in specificity, high in sensitivity, and convenient to operate; the warm starting TaqDNA polymerase can be applied to regular PCR amplification, real-time fluorogenic quantitative PCR, multiplex PCR, and digital PCR and TA cloning.

Description

technical field [0001] The invention relates to the fields of biochemistry and molecular biology, in particular to a hot-start Taq DNA polymerase and its preparation method and application. Background technique [0002] Taq DNA polymerase is a thermostable polymerase isolated from Thermus aquaticus. The nucleotide sequence of the coding region of the enzyme gene is 2496 bases, encoding 832 amino acids. It is a thermostable alkaline polymerase with a molecular weight of 94KD. Protein with DNA-binding ability. The good thermal stability of Taq DNA polymerase is the prerequisite for the enzyme to be used in PCR reactions, and it is also the main reason for the rapid development and wide application of PCR reactions. It is an essential tool enzyme in molecular biology. [0003] Before the denaturation of the first cycle of PCR, there will be some non-specific pairing between primers and templates during the heating process. If Taq DNA polymerase is active at this time, non-spec...

Claims

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Application Information

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IPC IPC(8): C12N9/12C12N15/54C12N15/70
CPCC12N9/1252C12N15/70C12N2800/22C12Y207/07007
Inventor 王丹宋璐赵世源蔡一荣曲俊杰徐桤李铁男赵倩李望丰谢占武
Owner 东北制药集团辽宁生物医药有限公司
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