Method for separating and detecting spontaneous mutation gene based on agarose gel denaturation and renaturation and biotin affinity adsorption

A technology of agarose gel and spontaneous mutation, which is applied in the direction of biochemical equipment and methods, microbiological determination/testing, etc., and can solve the problems of inability to solve the specific hybridization of enzyme-cut fragments and limit applications, etc.

Inactive Publication Date: 2013-01-02
POMOLOGY RES INST GUANGDONG ACADEMY OF AGRI SCI
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Problems solved by technology

However, the existing phenotypic cloning methods, such as genome subtraction, cannot solve the specific hybridization problem between enzyme-cut fragments, thus limiting their further application.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Identification and detection of mutation sites

[0038] 1. Extract the genomic DNA of Banana braziliana and Nongke No. 1 banana from the leaves.

[0039] 2. Take 5 μg wild-type genomic DNA (2.59x10 -17 mol) and 0.1 μg mutant genomic DNA (molecular weight of DNA = number of bases * 324.5), as the research object;

[0040] 3. Use an appropriate amount of Taq I restriction endonuclease to completely digest the above-mentioned genomic DNA (the wild-type genomic DNA is digested into about 62.16 pmol fragments, and the mutant genomic DNA is digested into about 1.24 pmol fragments);

[0041] 4. Connect the connector, the connector sequence is:

[0042] SEQ ID NO:1 5'-CATGCTTGTAGACTCACA-3'

[0043] SEQ ID NO:2 3'-ACGAACATCTGAGTGTGC-5'

[0044] The lower chain of the linker should use T4 polynucleotide kinase, and the reaction system should be determined according to the products of different companies. The ligation system is 50 μl, and the specific ...

Embodiment 2

[0052] Example 2 Sequencing of mutation sites

[0053] PCR amplification: PCR amplification was carried out using the above-mentioned solution recovered by adsorption as a template. The primer sequence was SEQ ID NO: 3: CATGCTTGTAGACTCACA, the annealing temperature was 55°C, and the extension time was 1.0 minutes; the electrophoresis results were detected by electrophoresis, and the amplification Amplify fragments for sequencing.

[0054] Sequencing results: Design primers according to the obtained sequences, amplify the genomic DNA of the above two respectively, sequence and compare the amplification results, and find that there is a variation in a NBS-LRR disease resistance gene fragment, the sequence is as follows (xxx represents deletion the sequence of):

[0055] Brazil plantain: SEQ ID NO: 4 gggaatgggt ggagttggta agaccactct ggcacgtxxx atattcaatg atgaaaggat

[0056] Nongke No. 1 banana: SEQ ID NO: 5 gggaatgggt ggagttggta agaccactct ggcacgtaaa atattcaatg atgaaaggat

[0...

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Abstract

The invention relates to a method for separating and detecting a spontaneous mutation gene based on agarose gel denaturation and renaturation and biotin affinity adsorption, which belongs to the technical field of biology, and relates to a method for separating and detecting a mutation gene. The method comprises the following steps of: marking wild deoxyribonucleic acid (DNA) fragments by using biotin, mixing the biotin-marked wild DNA fragments and DNA fragments to be detected, and performing denaturation and renaturation, wherein at the moment, DNA fragments which contain mutation sites cannot form heteroduplex DNA molecules because the biotin-marked wild DNA fragments with the same migration rate as the DNA fragments are not hybridized with the DNA fragments, DNA fragments which do notcontain the mutation sites are hybridized with the corresponding biotin-marked wild DNA fragments to form the heteroduplex DNA molecules, and the heteroduplex DNA molecules carry biotin marks; and adsorbing and recovering the molecules which carry the biotin marks by using streptavidin magnetic beads to separate the DNA fragments which contain the mutation sites. Compared with the conventional method, the method is simpler and more convenient and sensitive, can be used for accurately identifying and detecting the mutation sites, and has high reliability.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for separating spontaneously mutated genes based on agarose gel denaturation renaturation and biotin affinity adsorption. Background technique [0002] Bananas are the fourth largest food crop in the world after rice, wheat and corn. According to the statistics of the Food and Agriculture Organization of the United Nations (FAO), in the past 10 years, the world's banana planting area has generally shown an increasing trend. In 2007, the planting area was 66.1575 million mu, and the output reached 81.2634 million tons, a record high. In 2007, China's banana harvested area was 4.5825 million mu, ranking fifth in the world; the total output was 7.325 million tons, ranking second in the world. But the development of the industry is under the devastating threat of Fusarium wilt, the pathogen of Fusarium oxysporum f.sp. cubeense. Pathogens invade corms and pseudostems from banana ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 李春雨易干军孙清明
Owner POMOLOGY RES INST GUANGDONG ACADEMY OF AGRI SCI
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