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Establishment method of Pinctada martensii MSAP (methylation sensitive amplification polymorphism) technical system

A technology for pinnacle martensii and method establishment, which is applied in biochemical equipment and methods, microbe determination/inspection, etc., can solve problems that have not been reported in the research, and achieve low silver staining background, clear target bands, and easy Detection effect

Inactive Publication Date: 2015-05-20
SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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Problems solved by technology

Although a lot of related work has been carried out on the genetic improvement of Pinctada martensii, there have been no reports on the application of DNA methylation, an epigenetic regulation level, to molecular marker-assisted breeding.

Method used

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  • Establishment method of Pinctada martensii MSAP (methylation sensitive amplification polymorphism) technical system
  • Establishment method of Pinctada martensii MSAP (methylation sensitive amplification polymorphism) technical system

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Embodiment 1

[0027] 1. Extraction and quality detection of genomic DNA of Pinctada martensii

[0028] Mollusc DNA extraction kit (purchased from Omega Bio-Tek Company) was used to extract the genomic DNA of Pinctada martensii, and the selected tissue was adductor muscle of Pinctada martensii. Choose OD 260 / OD 280 The DNA between 1.7 and 1.9 was used as a double-enzyme-digested sample, and the total amount of each DNA sample used for double-enzyme digestion was 300ng.

[0029] 2. Double digestion of genomic DNA and ligation with primer adapters

[0030] Two DNA samples were subjected to double enzyme digestion reaction with EcoR Ⅰ and Msp Ⅰ, EcoR Ⅰ and Hpa Ⅱ, respectively. Optimize the enzyme digestion reaction time: two DNA samples were subjected to 0h, 2h, 4h, 6h, 8h, 10h and 16h enzyme digestion experiments to find the best enzyme digestion time, after digestion, warm at 70°C for 15 minutes to inactivate the enzyme. Two copies of digestion products were obtained respectively. The r...

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Abstract

The invention discloses an establishment method of a Pinctada martensii MSAP (methylation sensitive amplification polymorphism) technical system, which comprises the following steps: (1) extracting Pinctada martensii genome DNA (deoxyribonucleic acid); (2) performing double digestion on the genome DNA, and connecting with a primer linker; (3) establishing a pre-amplification reaction system and a reaction procedure; (4) establishing a selective amplification reaction system and a reaction procedure; and (5) performing denaturing polyacrylamide gel electrophoresis and silver staining color development. The establishment method of a Pinctada martensii MSAP technical system can provide an analysis foundation for DNA methylation genetic mechanism research of Pinctada martensii, DNA methylation regulation and control of gene expression and DNA methylation variation caused by external environment variation. The invention is suitable for germplasm analysis and identification of Pinctada martensii, development of DNA methylation molecular markers and correlation analysis of gene expression and DNA methylation.

Description

technical field [0001] The invention belongs to a method for establishing a molecular marker technology, and in particular relates to a method for establishing a technology system of Pinctada martensii MSAP. Background technique [0002] Pinctada martensii is the main species cultivated in Shanghai water pearls in the world, and it is widely distributed in the southern sea area of ​​my country. Due to the combined effects of long-standing unscientific breeding methods, disorderly culture in sea areas, and environmental stress, the germplasm resources of Pinctada martensii are degraded, and there are common problems such as slow growth of parent shellfish, easy disease, smaller individuals, and decline in pearl quality. . One of the main ways to solve the above problems is to genetically improve Pinctada martensii and to breed new species with strong disease resistance and stress resistance. In terms of the improvement of Pinctada martensii genetics and breeding, karyotype ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 李耀国何毛贤
Owner SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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