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Method for enhancing high GC content DNA fragment amplification efficiency by organic solvent

An organic solvent and amplification efficiency technology, applied in the field of molecular biology, can solve the problems of ineffectiveness and unclearness, and achieve the effect of convenient use, less side effects, and easy long-term storage at room temperature

Inactive Publication Date: 2009-03-11
TIANJIN UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

The above measures are effective for PCR amplification of some DNA templates with high GC content, but sometimes they are ineffective, and the reason is not clear

Method used

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  • Method for enhancing high GC content DNA fragment amplification efficiency by organic solvent
  • Method for enhancing high GC content DNA fragment amplification efficiency by organic solvent
  • Method for enhancing high GC content DNA fragment amplification efficiency by organic solvent

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Embodiment Construction

[0027] Below in conjunction with embodiment, the present invention is further described; Following embodiment is illustrative, not limiting, can not limit protection scope of the present invention with following embodiment.

[0028] The optimization method of the PCR technology involved in the present invention is realized by adding an organic solvent in the PCR system, and its specific operation method is as follows:

[0029] 1. Extraction of human genomic DNA to be amplified:

[0030] 2. Configuration and optimization of PCR system

[0031] According to the existing technology, the PCR system is different according to the different DNA fragments to be amplified. Specifically, the amount of dNTP, template, Mg2+, and primer added is selected according to different templates and different lengths of amplified fragments;

[0032] H in the PCR reaction system 2 O is double distilled water and above grade water, the amount of water added in the system should be adjusted accordi...

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Abstract

The invention relates to a method for strengthening the amplification efficiency of DNA segments with high GC content through an organic solvent, which comprises the following steps: firstly, extraction of DNA to be amplified; secondly, configuration and optimization of a PCR system; thirdly, setting and operation of PCR conditions; and fourthly, electrophoretic detection of PCR products, wherein the optimization of the PCR system is to add the organic solvent into the PCR reaction system; and the electrophoretic detection of the PCR products is to adopt 1 percent of sepharose gel, to dye by an EB liquid after glue leaking, to use an ultraviolet light-splitting transmissometer to observe the position of amplified segments in the gel, and to compare the amplified segments with corresponding molecular weight standards. The method has obvious effect of amplifying the DNA segments which are rich in GC alkaline base, so that the amplification of sequences with high GC content is more efficiently and has more purposefulness. The method has important theoretical significance and application value on development of the molecular biological technical tests.

Description

technical field [0001] The invention belongs to the polymerase chain reaction (PCR) amplification in the field of molecular biology, in particular to a method for enhancing the amplification efficiency of DNA fragments with high GC content by using an organic solvent as an optimization agent. Background technique [0002] PCR technology was developed on the basis of nucleic acid research. Since the concepts of DNA and RNA two nucleic acids were formally proposed in 1930, people have conducted more in-depth research on the chemical composition, structure and function of nucleic acids. In 1953, Watson The DNA double helix structure and its semi-conservative replication model proposed by Crisk and X-ray diffraction patterns and various chemical analysis data are a leap in the history of biological sciences, making great strides in gene cloning, expression, and regulation in vitro progress, and thus kicked off the prelude to genetic engineering. With the continuous research of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/34
Inventor 张治洲杨霞刘芳孟良玉
Owner TIANJIN UNIV OF SCI & TECH
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