Method for eliminating PCR amplification product pollution based on IIs type restriction endonuclease

A technology for restriction endonuclease and amplification product, which is applied in the field of eliminating PCR amplification product contamination based on type IIs restriction endonuclease, and can solve problems such as affecting PCR amplification.

Inactive Publication Date: 2014-06-11
HUADONG RES INST FOR MEDICINE & BIOTECHNICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The technical problem to be solved in the present invention is to use the IIs enzyme digestion method to eliminate PCR pollution, directly introduce the recognition site of the enzyme through the primer, the site needs to be close to the 3' end, and the mismatch at the 3' end of the primer will seriously affect the amplification of PCR. Therefore, we adopt a new type IIs restriction endonuclease, the cutting site is a base away from the recognition site, and establish a new anti-pollution method, which can effectively eliminate pollution without affecting the amplification efficiency

Method used

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  • Method for eliminating PCR amplification product pollution based on IIs type restriction endonuclease
  • Method for eliminating PCR amplification product pollution based on IIs type restriction endonuclease
  • Method for eliminating PCR amplification product pollution based on IIs type restriction endonuclease

Examples

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Embodiment 1

[0028] In order to investigate the elimination effect of this method on the product contamination after single-stranded template amplification, the single-stranded probe ligation product S-SRY was used as the single-stranded template, and the 10 μl ligation reaction system consisted of: genomic DNA extracted from human whole blood (100 ~ 200 ng), Probe 1 and Probe 2 mixture (5 fmol each), 1 U Ampligase, 20 mM Tris-HCl (pH 8.3), 25 mM KCl, 10 mM MgCl2, 0.5 mM NAD, 0.01% Triton X-100. The probe ligation conditions were pre-amplification at 94°C for 5 min, followed by 1 min at 94°C, and 4 min at 60°C, for a total of 10 cycles. The obtained ligation products were prepared by serial dilution after purification.

[0029] Design a pair of primers with the sequence of the single-stranded template S-SRY as the target sequence. The upstream and downstream primers match the sequence of the target gene, but the upstream primers are recognized by the FokI enzyme consisting of the recogniti...

Embodiment 2

[0039] In order to investigate the elimination effect of the present invention on the product contamination after double-stranded template amplification, the short nucleic acid fragment PPAR-N is used as the target double-stranded template, and a pair of primers are designed. The upstream and downstream primers match the sequence of the target gene, but the upstream primer introduces Mismatched bases recognized by type IIs restriction enzymes. The templates and primer sequences of Example 2 are shown in Table 3, where the underlined bases are mismatched bases.

[0040] Table 3. Example 2 primer sequence

[0041]

[0042] The total volume of the PCR reaction system is 25 μL, and the PCR blank reaction system without DNA template is 2 mmol / L MgCl 2 , 0.5mmol / L dNTP, 1×PCR Buffer (100mmol / L Tris-HCl, 500mmol / L KCl, 1% X-100), 1U Taq enzyme, 10 pmol primer, 0.4×SYBG dye, 0.6U FokI enzyme.

[0043] The double-stranded template PPAR-N enzyme digestion condition is 37°C for 40...

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Abstract

The invention belongs to the field of biotechnology and discloses a method for eliminating PCR amplification product pollution based on IIs type restriction endonuclease. When upstream primers and / or downstream primers are designed, a base sequence of IIs type restriction endonuclease recognition site is introduced. IIs type restriction endonuclease is added into a PCR reaction system. After enzyme digestion reaction of endonuclease, PCR amplification is directly carried out. If there is amplification product pollution in the PCR reaction system, IIs type restriction endonuclease can recognize the base sequence of IIs type restriction endonuclease recognition site on an amplification product, completely cut off a part complementary to the primers on the amplification product and make the amplification product unable to serve as a template of next round amplification, so as to eliminate pollution of polymerase chain reaction amplification product to PCR reaction. According to the method provided by the invention, pollution can be selectively eliminated without influencing normal amplification efficiency and specificity, and downstream operation of amplification product can be carried out normally.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a method for eliminating contamination of PCR amplification products based on type IIs restriction endonuclease. Background technique [0002] Polymerase chain reaction (PCR) is a highly sensitive nucleic acid amplification technology. However, the high sensitivity of PCR technology makes it very easy to contaminate and produce false positive results, which seriously interferes with the accuracy of PCR detection. The most important source of pollution during PCR amplification is the amplified product identical to the target sequence. High-concentration PCR products are easily splashed or form aerosols during the experimental operation. create pollution. [0003] Researchers at home and abroad have proposed many kinds of PCR anti-pollution technologies, including ultraviolet irradiation, acid hydrolysis, enzyme digestion, chemical modification and so on. Ultraviolet irradiatio...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10
Inventor 周国华王璐茜
Owner HUADONG RES INST FOR MEDICINE & BIOTECHNICS
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