Preparation method and application of uracil DNA glycosidase

The technology of uracil and glycosidase is applied in the field of preparation of uracil DNA glycosidase, which can solve the problems such as the inability of the amplification reaction to be carried out effectively, the reduction of the yield of the DNA amplification reaction, the reduction of detection sensitivity, etc. The effect of improving accuracy and sensitivity and improving detection sensitivity

Inactive Publication Date: 2017-05-31
SUZHOU KUANGSHI JUNCHI BIOLOGICAL SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The previous uracil DNA glycosidase has high stability at high temperature and is not easy to be inactivated, which leads to the hydrolysis of dU in the synthesized DNA, thereby fragmenting the DNA, making the amplification reaction ineffective, resulting in DNA amplification. The output of the amplification reaction is greatly reduced, which reduces the detection sensitivity

Method used

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  • Preparation method and application of uracil DNA glycosidase
  • Preparation method and application of uracil DNA glycosidase
  • Preparation method and application of uracil DNA glycosidase

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Example 1 Preparation of uracil DNA glycosidase from psychrophilic bacteria Colwellia psychrerythraea

[0031] The first step is to design and synthesize the uracil DNA glycosidase gene of psychrophilic bacteria Colwellia psychrerythraea, and insert it into the pET28 expression vector to construct the recombinant expression plasmid of uracil DNA glycosidase. The N-terminus of the recombinant psychrophilic bacterium Colwelliapsychrerythraea uracil DNA glycosidase has six consecutive histidine affinity purification tags derived from the pET28 vector, which is used for immobilized nickel ion affinity chromatography purification.

[0032] The second step is to recombinantly express uracil DNA glycosidase from psychrophilic bacteria Colwellia psychrerythraea. The psychrophilic bacterium Colwellia psychrerythraea uracil DNA glycosidase recombinant expression plasmid was transformed into Escherichia coli expression host BL21 (DE3), and the psychrophilic bacterium Colwellia psy...

Embodiment 2

[0037] Example 2 Application of psychrophilic bacteria Colwellia psychrerythraea uracil DNA glycosidase as a PCR contamination remover

[0038] Utilization of purified psychrophilic bacterium Colwellia psychrerythraea uracil DNA glycosidase as a contamination scavenger for Pfu DNA polymerase-catalyzed PCR. The target gene for amplification is Escherichia coli DNA polymerase IV gene, PCR reaction buffer: 20 mM Tris-HCl (pH 8.8), 10 mM (NH4)2SO4, 10 mM KCl, 0.1 mg / mL BSA, 0.1% (v / v) Triton X-100, 2 mM MgSO4; Other components are 0.2 mM dNTP, 0.3 μM primer, 20ng Escherichia coli DNA polymerase IV gene fragment (including dU base), 2.5 units of Pfu DNA polymerase. The agarose gel electrophoresis pictures of PCR amplification results are shown in image 3 , the results show that the psychrophilic bacteria Colwellia psychrerythraea uracil DNA glycosidase can effectively eliminate DNA pollutants containing dU bases.

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Abstract

The invention discloses a preparation method and an application of uracil DNA glycosidase. The uracil DNA glycosidase prepared by using the method can be used for hydrolyze glucosidic bonds between uracil basic groups and deoxyriboses in DNA at the temperature of 20-37 DEG C; at the same time the glycosidase has a good heat sensitivity, when the temperature is higher than 60 DEG C, the glycosidase swiftly loses activeness, when the temperature reaches 60 DEG C for 5 minutes, the enzymatic activity loses by 99%; the prepared heat sensitive uracil DNA glycosidase can be used for preventing the sample DNA contamination in all types of nucleic acid detection reaction. The preparation method and application of uracil DNA glycosidase have the advantages being capable of both completely removing the contaminated DNA, and exerting no inhibition to the nucleic acid amplified reaction. The glycosidase can be used as a cleaning agent to the sample contamination in all types of nucleic acid amplified reaction (such as PCR), can remove the DNA contamination caused by the products of the previous amplified reaction, and increase the accuracy of nucleic acid test.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a preparation method and application of uracil DNA glycosidase. Background technique [0002] The emergence of PCR technology has made it possible to quickly prepare specific DNA molecules in vitro, and is currently widely used in various nucleic acid amplification and detection fields, which has greatly promoted the development of nucleic acid-based molecular diagnostic techniques. In molecular diagnostics based on PCR and other nucleic acid amplification techniques, the cross-contamination of DNA samples, especially the contamination of subsequent test samples by previous test samples, directly affects the accuracy of test reactions. In order to eliminate cross-contamination of different batches of DNA samples, dUTP is added to the PCR reaction system so that the synthesized DNA contains dU bases, and uracil DNA is added to the samples before the next PC...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12N15/70C12Q1/68
CPCC12N9/2497C12N15/70C12N2800/22C12Q1/6848C12Y302/02003C12Q2531/113
Inventor 刘喜朋
Owner SUZHOU KUANGSHI JUNCHI BIOLOGICAL SCI & TECH
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