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SNP (Single Nucleotide Polymorphism) classification method and application based on PCR (Polymerase Chain Reaction)

A typing method and base technology, applied in the field of molecular biology and bioinformatics, can solve the problems of cumbersome experimental process, long time consumption, harsh experimental conditions and other problems of CAPs method, and achieve controllable throughput, simple operation, Detect easy effects

Active Publication Date: 2013-09-11
INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
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Problems solved by technology

However, the above methods have certain disadvantages: SSCP can only use non-denatured glue, and the experimental conditions are harsh, requiring room temperature conditions, which are difficult to achieve in general laboratories; the experimental process of CAPs is cumbersome, and an experimental procedure takes a long time; MassARRAY mass spectrometry The cost of TaqMan probe method and other chip methods is quite high, and it is difficult for ordinary laboratories to afford

Method used

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  • SNP (Single Nucleotide Polymorphism) classification method and application based on PCR (Polymerase Chain Reaction)
  • SNP (Single Nucleotide Polymorphism) classification method and application based on PCR (Polymerase Chain Reaction)
  • SNP (Single Nucleotide Polymorphism) classification method and application based on PCR (Polymerase Chain Reaction)

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Embodiment 1

[0042] A PCR-based SNP typing method, its steps are:

[0043] 1. Use CTAB method to extract total DNA from leaves:

[0044] A. After taking out the appropriate amount of Brassica napus No. 11 and 73290 leaf samples from the ultra-low temperature refrigerator (-70℃), immediately put them into a frozen mortar, add liquid nitrogen and grind into powder; quickly put it into 50ml centrifuge Into the tube, add the extract (0.2M Tris-Cl, 0.25NaCl, 25mM EDTA, 0.5% (mass ratio) SDS, pH 7.5) preheated in a 60℃ water bath, mix well, and put it in the 60℃ 40min in the water bath;

[0045] B. Take out the centrifuge tube, add an equal volume of chloroform: isoamyl alcohol (24:1, V / V), slowly invert the centrifuge tube up and down 30-50 times to mix well, and centrifuge at 1300g for 10 minutes;

[0046] C. Take the supernatant into another centrifuge tube, add an equal volume of chloroform: isoamyl alcohol (24:1, V / V), and extract again. Take the supernatant, add 0.6 times volume of ice-cold isoa...

Embodiment 2

[0070] Traditional molecular breeding methods for crops aggregate multiple excellent traits and often use SSR markers. The density of this marker in the genome is low, and it is difficult to find markers that closely linked functional genes. The application of the labeling method of the present invention can achieve the degree of linkage that cannot be achieved by SSR labeling, and greatly improve the efficiency. Existing materials are Shuang 11 (Guo Shenyou 2008030) and 73290 materials. Among them, Zhong Shuang 11 has excellent properties such as crack angle resistance, lodging resistance, anti-bacterial sclerosis, long siliques, and a thousand grains, while 73290 has branches. Good traits such as many, many siliques in the whole plant. The current goal is to transfer the excellent traits of 73290 material with more branches and more siliques into the Zhongshuang 11 material to achieve the goal of improving Zhongshuang 11.

[0071] The application of a PCR-based SNP typing meth...

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Abstract

The invention provides an SNP (Single Nucleotide Polymorphism) classification method and application based on PCR (Polymerase Chain Reaction). The SNP classification method comprises the steps of: (1) selecting a plant sample for experiment-tissues of a plurality of strains of certain species; (2) extracting genomic DNA of the tissues; (3) purifying the genomic DNA as a template for PCR amplification; (4) selecting AT type SNP in an SNP database of the species; (5) screening out SNP of which the third base before the SNP locus is G; (6) using primer 3 software to design a primer so that the last base of a positive primer is at the SNP locus and the last base is T; (7) introducing mispairing, and changing the antepenultimate base of the positive primer into A; (8) performing PCR amplification; (9) treating the PCR products by means of modified polyacrylamide gel electrophoretic separation; and (10) observing whether amplification band exists. The invention further provides application of the SNP classification method based on PCR in pyramiding breeding; the method is easy to implement, simple, convenient, and low in cost; the detection is simple and efficient; the classification success rate of SNP is as high as 91%.

Description

Technical field [0001] The invention relates to the fields of molecular biology and bioinformatics, in particular to a method for SNP typing by PCR, and also relates to the application of a PCR-based SNP typing method in rape polymerization breeding. The method is fast, simple, and low in cost, and can be widely used in fields such as disease diagnosis and crop breeding. Background technique [0002] Molecular markers are genetic markers based on the variation of nucleotide sequences in genetic material between individuals, and are a direct reflection of genetic polymorphisms at the DNA level. Compared with several other genetic markers-morphological markers, biochemical markers, and cytological markers, DNA molecular markers have the following advantages: most molecular markers are co-dominant, and it is very convenient to select recessive traits; Genome variation is extremely rich, and the number of molecular markers is almost unlimited; at different stages of biological devel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 华玮黄顺谋王汉中师家勤刘胜毅
Owner INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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