Method for sifting macrobrachium nipponensis microsatellite mark with magnetic bead concentration method

A microsatellite marker and microsatellite technology, applied in the field of Macrobrachium japonicus DNA molecular genetic markers, can solve the problems of harm to the human body, high radioactivity, and difficulty in popularization, and achieve the effects of high reliability, low cost, and short cycle

Inactive Publication Date: 2009-04-08
FRESHWATER FISHERIES RES CENT OF CHINESE ACAD OF FISHERY SCI
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Problems solved by technology

Although the isotope sensitivity is high, it is difficult to fully promote due to the high radioactivity, which requires high laboratory protection and is harmful to the human body.

Method used

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Embodiment Construction

[0046] 1. Extract the genomic DNA of Macrobrachium japonicus, the method is as follows:

[0047] Take about 200 mg of live Macrobrachium japonicus muscle tissue in a 1.5 mL Eppendorf tube, cut it into pieces, add 665 μl of extraction buffer (Tris.Cl: 10 mM, pH=8.0; EDTA: 0.1 mM, pH=8.0), and then add 35 μl of SDS (0.5 %) and 10 μl of proteinase K (20 mg / mL), digested overnight in a water bath at 55°C. Add an equal volume of saturated phenol (700 μl) and mix slowly with a DNA mixer for 30 minutes, then centrifuge at 12,000 rpm for 10 minutes in a refrigerated centrifuge and extract once. Add an equal volume of chloroform / isoamyl alcohol (24:1) (700 μl) to the supernatant, mix slowly with a DNA mixer for 15 minutes, and then extract once by centrifuging in a refrigerated centrifuge at 12,000 rpm for 15 minutes. The supernatant was added to an equal volume of chloroform (700 μl), mixed slowly by a DNA mixer for 10 minutes, and then extracted once by centrifugation in a refrigera...

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Abstract

The invention provides a method for screening microsatellite markers of Macrobrachium nipponensis by a magnetic beads enrichment method, including the following steps: the extraction of the genomic DNA of the Macrobrachium nipponensis; the hybridization of a probe and a target segment; the enrichment of magnetic beads; positive sequencing clone and the amplification of the genomic DNA according to primers which are related to the flanking sequences of the microsatellite. As the invention combines the magnetic beads enrichment and PRC amplification of bacterial liquid to carry out second-time screening, no isotope is needed. The method has the advantages of safety and high efficiency and low cost, short cycle, high reliability and the like. At the same time, in the enrichment process, the magnetic beads are repeatedly washed for a plurality of times, which can clean and remove microsatellite sequences which are not firmly adhered because of less repetition times. Therefore, the obtained microsatellite sequences are longer and are repeated for more times. The microsatellite sequences which are more frequently repeated are considered to have rich diversity at interspecies and intraspecies and can be widely applied to the diversity identification of germ plasm, the building of genetic maps, and QTL. Therefore, the method is one which screens the microsatellite markers of Macrobrachium nipponensis and can be easily popularized.

Description

technical field [0001] The invention belongs to a DNA molecular genetic marker method of Macrobrachium japonicus, in particular to a method for rapidly separating and screening microsatellite markers of Macrobrachium japonicus by using a magnetic bead enrichment method. Background technique [0002] Macrobrachium nipponensis (Macrobrachium nipponensis), commonly known as freshwater shrimp, belongs to the class Crustacea, Decapod order, Longarm shrimp family, and Macrobrachium genus. It has the characteristics of strong adaptability, wide distribution, miscellaneous food habits, fast growth, and high economic benefits of breeding. It is an important cultured species of freshwater shrimp in my country. Most of the current research work on Macrobrachium japonicus focuses on growth characteristics (Liu Jun et al., 2003), karyotype analysis (Yang Wanxi and Zhou Hong, 2000) and seedling cultivation (He Xugang and Gong Shiyuan, 2003). Due to the limited genetic background data of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 傅洪拓李法君吴滟龚永生
Owner FRESHWATER FISHERIES RES CENT OF CHINESE ACAD OF FISHERY SCI
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