PCR pollution prevention method and applications thereof

A technology of reaction solution and reaction system, applied in the field of biotechnology and molecular biology, to achieve the effect of increasing difficulty and complexity, eliminating pollution sources, and simple and easy experimental operation

Inactive Publication Date: 2017-01-18
TINYGENE BIOTECH SHANGHAI CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] One of the technical problems to be solved by the present invention is to provide a method for preventing PCR pollution, which can overcome the problem of exogenous microbial DNA or PCR product aerosol pollution that occurs during the PCR amplification process, and does not affect the PCR amplification efficiency. It will not increase the difficulty and cumbersomeness of subsequent molecular experiments

Method used

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  • PCR pollution prevention method and applications thereof
  • PCR pollution prevention method and applications thereof
  • PCR pollution prevention method and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1B

[0031] Embodiment 1 Benzonase nuclease carries out pretreatment to PCR reaction system

[0032] Benzonase nuclease (purchased from Merck company) final concentration is 10 -8 U / μL~10 -1 U / μL, when used, add Benzonase nuclease to the conventional PCR reaction system (see Table 2) that removes primers and templates, divide the processed system into 96-well plates, and perform subsequent processing in a PCR machine .

[0033] Table 1 Conventional PCR reaction system

[0034] Element concentration Taq DNA polymerase 5U 10× enzyme buffer 5μL dNTPs 100μmol / L template DNA 1ng-100ng forward primer 200nM reverse primer 200nM double distilled water Make up to 50μL

[0035] Table 2 has removed the conventional PCR reaction system of primer and template

[0036] Element concentration Taq DNA polymerase 5U 10× enzyme buffer 5μL dNTPs 100μmol / L double distilled water Make up to 47μL ...

Embodiment 2

[0044] Embodiment 2: the application of the present invention in common qualitative PCR

[0045] The PCR reaction system treated with Benzonase nuclease (see Example 1) and the conventional PCR reaction system (that is, the PCR reaction system not treated with Benzonase nuclease) were used to amplify the 16S V3-V4 region of the sample by PCR, 33 cycles , the electrophoresis detection results are as follows figure 1 shown.

[0046] The results of electrophoresis showed that when the PCR amplification reached 33 cycles, the negative control of the conventional PCR reaction system had obvious bands, but the negative control of the PCR reaction system treated with Benzonase nuclease could not detect any bands at all.

[0047] From the results of electrophoresis, we can preliminarily know that the enzyme treatment system can remove the background pollution caused by pollution sources such as system and aerosol, but if the enzyme treatment system is applied to high-throughput techn...

Embodiment 3

[0048] Example 3: Application of the present invention in high-throughput sequencing molecular library construction

[0049] Select A sample, and use the PCR reaction system treated with Benzonase nuclease (see Example 1) and the conventional PCR reaction system (that is, the PCR reaction system without Benzonase nuclease treatment) to perform PCR on the V3-V4 region of its 16S rRNA gene For amplification, the target band can be detected in the amplified sample but the negative control cannot detect the band as the standard, and this sample is used as the positive control. When the cycle number increased, the negative control could detect the corresponding target band, but the negative control of the treatment system could not detect the target band. The results of electrophoresis were as figure 2 shown.

[0050] The above 4 samples (such as figure 2 A, NA, TA, N--) in Illumina Miseq high-throughput sequencing, statistical analysis of optimized sequences after quality scr...

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Abstract

The invention discloses a PCR pollution prevention method. The method comprises the following steps: 1, adding a certain amount of Benzonase nuclease into a routine PCR system free of primers and templates to make the Benzonase nuclease play a role in enzyme digestion at a proper temperature in order to completely digest DNA polluted by exogenous microbes or aerosols existing in the PCR system; 2, keeping the Benzonase nuclease treated PCR system at a high temperature to inactivate the Benzonase nuclease in a reaction solution; and 3, adding corresponding primers and templates, and carrying out subsequent PCR amplification. The invention also discloses applications of the method in common qualitative PCR, fluorescent quantitative PCR and high-flux sequencing molecule database creation. The method effectively solves the exogenous microbe DNA or PCR product aerosol pollution problem appearing in the PCR amplification process, and has wide application prospect in high-flux sequencing.

Description

technical field [0001] The invention relates to a method for preventing PCR pollution and application thereof, belonging to the field of biotechnology and molecular biology. Background technique [0002] PCR is a very common method for amplifying and enriching target DNA fragments in molecular biology, with extremely high sensitivity. However, contamination of the PCR reaction often occurs, resulting in false positive results, which is the biggest problem facing the PCR method. The high-throughput sequencing that has been popular in recent years can detect trace microorganisms in the environment by obtaining a large amount of sequence information. However, microorganisms are everywhere in the environment. If there is no good prevention of residual microbial genomic DNA in the PCR amplification system , PCR product aerosol and other background nucleic acid pollution methods produced in previous experiments will have a certain degree of impact on our experiments and data anal...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12Q1/6848C12Q2521/301
Inventor 丁旭候美玲韩海燕
Owner TINYGENE BIOTECH SHANGHAI CO LTD
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