Method for processing DNA mould and improving DNA expansion efficiency by using restriction enzymes

A technology of restriction endonuclease and amplification efficiency, applied in the field of using restriction endonuclease to treat DNA template to improve DNA amplification efficiency, to solve the problem of low PCR amplification efficiency, obvious amplification effect, and difficult amplification. increased effect

Inactive Publication Date: 2008-01-30
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] In recent years, the development of biotechnology is changing with each passing day. Literature research and patent retrieval have found that there has been no corresponding research on the method of using restriction endonucleases to treat DNA templates to improve the efficiency of polymerase chain reaction (PCR) amplification.

Method used

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  • Method for processing DNA mould and improving DNA expansion efficiency by using restriction enzymes
  • Method for processing DNA mould and improving DNA expansion efficiency by using restriction enzymes
  • Method for processing DNA mould and improving DNA expansion efficiency by using restriction enzymes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] After digestion with BamHI, BglII, EcoRI, and EcoRV, the amplified fragment is a DNA fragment of 754bp with a GC content of 61%.

[0053] 1. Configuration of PCR reaction system:

[0054] Configure the system in a thin-walled PCR tube in the following order and dosage.

[0055] 10×PCR buffer (with Mg 2+ ) 2.5μL

[0056] dNTP substrate (2.5mM) 2.5μL

[0057]Primer 1 (2.0μM) 2μL

[0058] Primer 2 (2.0μM) 2μL

[0059] Template 2 μL

[0060] Taq enzyme (5U / μL) 0.5μL

[0061] h 2 O up to 25μL

[0062] Among them, the human Genomic DNA template was digested by the above-mentioned various endonucleases, wherein the endonuclease was purchased from Canada BBI Company, and the Taq enzyme was purchased from Beijing Sanbo Yuanzhi Bioengineering Company.

[0063] A total of 5 PCR system tubes were configured, numbered from 0 to 4.

[0064] 2. The templates added sequentially from the 0th to the 4th tubes are undigested and digested with BamHI, BglII, EcoRI, and EcoRV.

[...

Embodiment 2

[0073] The amplified fragments were 790bp DNA fragments with a GC content of 78% after single digestion with BamHI and XhoI respectively.

[0074] 1. Configuration of PCR reaction system:

[0075] Configure the system in a thin-walled PCR tube in the following order and dosage.

[0076] 10×PCR buffer (with Mg 2+ ) 2.5μL

[0077] dNTP substrate (2.5mM) 2.5μL

[0078] Primer 1 (2.0μM) 2μL

[0079] Primer 2 (2.0μM) 2μL

[0080] Template 2 μL

[0081] Taq enzyme (5U / μL) 0.5μL

[0082] h 2 O up to 25μL

[0083] Among them, the human Genomic DNA template was digested by the above-mentioned various endonucleases, wherein the endonuclease was purchased from Canada BBI Company, and the Taq enzyme was purchased from Beijing Sanbo Yuanzhi Bioengineering Company.

[0084] A total of 2 PCR system tubes were configured, and numbered from 1 to 2.

[0085] Note: The template has not been digested, and various measures (optimizing conditions, adding synergists, etc.) still cannot be ...

Embodiment 3

[0095] After the templates were digested with XbaI, EcoRI, and EcoRI+XhoI, the amplified fragments were 1541bp DNA fragments with a GC content of 75%.

[0096] 1. Configuration of PCR reaction system:

[0097] Configure the system in a thin-walled PCR tube in the following order and dosage.

[0098] 10×PCR buffer (with Mg 2+ ) 2.5μL

[0099] dNTP substrate (2.5mM) 3μL

[0100] Primer 1 (2.0μM) 2.5μL

[0101] Primer 2 (2.0μM) 2.5μL

[0102] Template 2 μL

[0103] Taq enzyme (5U / μL) 0.5μL

[0104] Pfu enzyme (2.5U / μL) 0.5μL

[0105] h 2 O up to 25μL

[0106] Among them, the human Genomic DNA template template is digested by the above-mentioned various endonucleases, wherein the endonucleases are purchased from Canada BBI Company, and Taq enzyme and Pfu enzyme are purchased from Beijing Sanbo Yuanzhi Bioengineering Company.

[0107] A total of 4 tubes of the PCR system were configured and numbered from 0 to 3.

[0108] Note: The template has not been digested, and vari...

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PUM

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Abstract

The invention relates to a method by using limited endonuclease to treat a DNA template to increase amplification efficiency of polymerase chain reaction. The method adopts limited endonuclease to treat the DNA template and realizes effective amplification of the DNA fragment which is difficult to be amplified through enzyme endonuclease to the DNA template to be amplified, thereby effectively improving the amplification of the DNA fragment, more particularly of the fragment containing high GC content, and having the advantages of remarkable effect and simple use and operation and better solving key problems that certain DNA fragment has low amplification efficiency or difficult amplification.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, in particular to a method for improving DNA amplification efficiency by using restriction endonuclease to treat DNA templates. Background technique [0002] Polymerase chain reaction (PCR), also known as in vitro enzymatic gene amplification, is a gene amplification technology that simulates DNA replication in vitro. This technology was invented by K.Mullis in 1985. Under the action of enzymes and the guidance of specific primers, a large number of gene duplications that only occur when organisms cell division and proliferation can be realized in a test tube in a very short period of time. Generally speaking, within a few hours. Amplify the gene of interest to a million-fold. [0003] Over the past 20 years, PCR technology has gradually matured through continuous progress and development, forming a complete set of technical systems. Conventional PCR technology is easy to operate and h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12Q1/68
Inventor 孟良玉李静阎大伟张治洲
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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