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Method for establishing and optimizing exopalaemon modestus SRAP(sequence-related amplified polymorphism)-PCR(polymerase chain reaction) system

A technology of SRAP-PCR and optimization method, which is applied in the field of establishment and optimization of SRAP-PCR system of white shrimp, can solve the problems of large error in experimental results, cumbersome process, and it is difficult to take into account the interaction and other problems.

Inactive Publication Date: 2011-11-09
FRESHWATER FISHERIES RES CENT OF CHINESE ACAD OF FISHERY SCI
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Problems solved by technology

This molecular marker technology has the advantages of simplicity, rapidity, stability, and no need to predict sequence information. It is not only widely used in plant genetic diversity analysis, gene mapping, and genetic map construction. It has been used in Macrobrachium rosenbergii and Macrobrachium japonicus in recent years. , Macrobrachium hainanensis, grass carp, bream and other aquatic animal genetic diversity research; in the optimization of the reaction system, in the past, simple gradient experiments were often used to explore the best levels of each factor one by one, the process was cumbersome, and It is difficult to take into account the interaction between various factors, and the experimental results have large errors

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  • Method for establishing and optimizing exopalaemon modestus SRAP(sequence-related amplified polymorphism)-PCR(polymerase chain reaction) system
  • Method for establishing and optimizing exopalaemon modestus SRAP(sequence-related amplified polymorphism)-PCR(polymerase chain reaction) system

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Embodiment 1

[0019] The present invention is a method for establishing and optimizing the SRAP-PCR system of the beautiful white shrimp, which adopts the following process steps:

[0020] 1. Collect a sample of the white shrimp, save it in 95% ethanol and bring it back to the laboratory. The total DNA of the samples was extracted by conventional phenol / chloroform method. The extracted DNA was washed with 70% ethanol, dried at room temperature, dissolved in an appropriate amount of ddH20, and stored at -20°C for future use.

[0021] 2. Synthesize SRAP primer sequences with reference to relevant literature, select some white shrimp samples for pre-amplification, and screen primer pairs with good amplification effects. The sequence of the screened SRAP primer pair is:

[0022] ME2 (5′-TGAGTCCAAACCGGAGC-3′) and

[0023] EM4 (5'-GACTGCGTACGAATTTGA-3').

[0024] 3. Factors and level gradient design of PCR reaction, as shown in Table 1.

[0025] Table 1 Factors and levels of PCR reaction

...

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Abstract

The invention relates to a method for establishing and optimizing an exopalaemon modestus SRAP(sequence-related amplified polymorphism)-PCR(polymerase chain reaction) system, belonging to the technical field of fisheries biology. The method for establishing and optimizing an exopalaemon modestus SRAP-PCR system is characterized in that 25mu L of the PCR system contains 2mmol / L of Mg<2+>, 0.5U of Taq enzyme, 75ng of template DNA (deoxyribonucleic acid), 0.20mmol / L of dNTPs and 0.2mu mol / L of primers, wherein the primers are 5'-TGAGTCCAAACCGGAGC-3' and 5'-GACTGCGTACGAATTTGA-3'. The currently reported molecular markers applied to exopalaemon modestus blastogensis analysis are only RAPD (randomly amplified polymorphic DNA) and AFLP (amplified fragment length polymorphism). The invention develops a new molecular marker (SRAP molecular marker), and supplements and enriches the means and methods for molecular biological researches on exopalaemon modestus.

Description

technical field [0001] The present invention relates to a method for establishing and optimizing the SRAP-PCR system of the white shrimp, which can quickly and accurately establish the optimized SRAP-PCR reaction system of the white shrimp, and the genetic diversity of the white shrimp can be developed conveniently by means of this method and germplasm resource investigation, belonging to the field of fishery biotechnology. Background technique [0002] The beautiful white shrimp (Exopalaemon modestus) belongs to Crustacea, Decapoda, Cyprididae, and White Shrimp. It is widely distributed in freshwater lakes and rivers in my country. It is the dominant species of shrimp in many medium and large lakes in the middle and lower reaches of the Yangtze River. Taihu Xiuli White Shrimp is called "Taihu Three Treasures" together with whitebait and plum anchovy because of its thin shell, delicious taste, rich nutrition and high yield. Zehu's "humi" (shrimp from white shrimp) is sold a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/10
Inventor 张敏莹施炜纲徐东坡刘凯段金荣程长洪
Owner FRESHWATER FISHERIES RES CENT OF CHINESE ACAD OF FISHERY SCI
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