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Method for establishing technical analysis system of prawn MSAP (methylation sensitive amplification polymorphism)

A technology for technical analysis and establishment of methods, applied in the field of molecular marker establishment, can solve problems such as inability to study adaptability, and achieve the effects of clear methylation sites, high polymorphism, and low cost

Inactive Publication Date: 2012-10-24
YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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Problems solved by technology

[0004] For the problems existing in the related technology, the purpose of the present invention is to provide a kind of MSAP technical system applicable to the DNA methylation analysis of the Chinese prawn genome, to provide a basis for studying the epigenetic adaptation mechanism of the Chinese prawn genome DNA methylation, to solve Unable to study the adaptability of Chinese shrimp through DNA methylation

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  • Method for establishing technical analysis system of prawn MSAP (methylation sensitive amplification polymorphism)

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[0020] The technical method of the present invention is described in detail below through the examples: first extract the Chinese prawn genomic DNA, and dilute it for later use; The reaction system of amplification and selective amplification was amplified by PCR; the amplified products were subjected to polyacrylamide gel electrophoresis and silver staining to obtain the desired bands.

[0021] (1) Extraction of Chinese shrimp genome DNA. Muscle DNA was extracted using the traditional saturated phenol-chloroform method. DNA samples with a purity of OD260 / OD280 between 1.8 and 1.9 were selected for quantification, diluted to a concentration of 200ng / μL, and stored at -20°C until use.

[0022] (2) Enzyme digestion of genomic DNA. In order to determine the optimal double digestion amount and digestion time, Hpa II / EcoR I and Msp I / EcoR The double enzyme digestion amount of I was set to 5, 7.5 and 10 U, and the double enzyme digestion time was set to 6, 8 and 10 h, r...

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Abstract

The invention provides a method for establishing a technical analysis system of prawn MSAP (methylation sensitive amplification polymorphism). The method includes: firstly, extracting prawn genome DNA (deoxyribonucleic acid), and diluting for standby; secondly, establishing a double digestion system and a ligation system; thirdly, establishing a pre-amplification reaction system and a pre-extension reaction program; fourthly, establishing a selective-extension reaction system and a reaction program; and fifthly, performing polyacrylamide gel electrophoresis and silver stain to obtain a required stripe. The method provides basis for search on methylation and epigenetic adaptive mechanism of Fenneropenaeus chinensis genome DNA and solves the problem that adaptation of Fenneropenaeus chinensis cannot be researched by DNA methylation. The method is applicable to germplasm resources identification, gene expression regulation and genetic marking of Fenneropenaeus chinensis, has the advantages of high polymorphism, low cost and clear methylation sites, and can be used to analyze methylation level of whole genome without knowing DNA sequence in advance.

Description

technical field [0001] The invention belongs to a method for establishing molecular markers, and in particular relates to a DNA methylation analysis method for Chinese prawn genome, and a method for establishing a prawn MSAP technical analysis system. Background technique [0002] DNA methylation is one of the earliest discovered methods of gene epigenetic modification. It is a modification method that regulates genomic DNA at the transcriptional level. It participates in processes such as gene expression, growth and development, and stress response. important role in regulation. The most commonly used technology for the study of genome methylation is methylation sensitive amplified polymorphism (Methylation sensitive amplified polymorphism, MSAP), which uses the isoschizase that recognizes the 5'-CCGG sequence Hpa II and Msp Ⅰ The sensitivity to genomic DNA methylation is different. The same sequence can amplify different bands to detect methylation levels and effectivel...

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 何玉英李健王清印杜盈
Owner YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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