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Process for fast separating, screening microsatellite mark of sea shell kind

A microsatellite marker, shellfish technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, and microbial determination/inspection, which can solve the waste of manpower and material resources, insufficient sequence information to meet the needs of screening, no Sequence information, etc.

Inactive Publication Date: 2006-06-28
OCEAN UNIV OF CHINA
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AI Technical Summary

Problems solved by technology

Therefore, the traditional screening method has caused a great waste of manpower and material resources
In recent years, with the development of computers and information networks, it has become a simple and cheap way to screen microsatellite DNA from sequences published in public databases. Insufficient sequence information for screening or no sequence information at all
[0004] Different screening methods and their efficiency have become the focus of scholars' attention and research. Different scholars have also tried different screening methods and strategies to improve screening efficiency. Different scholars and laboratories at home and abroad have also reported the hybrid screening method ( Hybridization Selection), FIASCO (Fast Isolation by AFLP of Sequences Containing Repeats) method and RAPD—Microsatellite probe Southern hybridization method and other methods, but in the application of shellfish, there are common shortcomings: the positive clone rate is low, and the efficiency is relatively low

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  • Process for fast separating, screening microsatellite mark of sea shell kind
  • Process for fast separating, screening microsatellite mark of sea shell kind

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Embodiment Construction

[0010] 1. Extract the genomic DNA of shellfish, the method is as follows:

[0011] After shellfish biopsy, take about 0.1 g of adductor muscle, add 500 μl of STE lysis buffer (NaCl: 100 mM; EDTA: 1 mM, pH=8.0; Tris-HCl, 10 mM, pH=8.0), cut it into pieces, and then add 50 μl of SDS (10%), and 5 μl of proteinase K (20 mg / ml), treated at 56° C. until the lysate was clear. Add an equal volume of saturated phenol (250 μl), chloroform / isoamyl alcohol (24:1) (250 μl), and extract 3 times. Take the supernatant, add an equal volume of chloroform / isoamyl alcohol (500 μl) and extract once. Take the supernatant, add 50 μl NaAc (3M), shake slowly, add ice absolute ethanol, and centrifuge at 12000 rpm for 10 min. Nucleic acids are precipitated at the bottom of the tube. The precipitate was washed with 70% 7 alcohol (1000 μl) and dried until all the ethanol evaporated. Add 100 μl of sterile water and a small amount of RNase A, and store in a 4°C refrigerator.

[0012] 2. The constructio...

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Abstract

The invention relates to a method used to quickly separate and screen seashell micro satellite sign. It includes the following steps: extracting seashell genome DNA; preserving in refrigerator at 4 centigrade degree; utilizing the extracted DNA to gain seashell genome DNA segment by existing AFLP method or restriction enzyme method and building enrichment library; crossing escherichia coli colony home position and confirming positive cloning; sequencing and design primer amplification genome DNA. The method has good reliability and repeatability. And its positive cloning efficiency can reach 100%. Once screening can gain hundreds sites. And a lot of effective technique method of micro satellite signs can be gained in one week.

Description

Technical field: [0001] The invention belongs to shellfish DNA molecular genetic marker technology, in particular to a technical method for rapidly separating and screening shellfish microsatellite markers. Background technique: [0002] The rapid development of molecular marker technology in recent years, especially the wide application of PCR-based molecular markers, provides a powerful tool for molecular genetics research on individual genetic characteristics and population genetic structure, among which the representative marker system— — Microsatellite markers are becoming more and more popular. Microsatellites, also known as simple tandem repeats (Simple Sequence Repeats, SSRs), refer to DNA sequences repeated in tandem multiple times in units of a few nucleotides (mostly 1 to 6). Because microsatellite DNA widely exists in the genome, and has the advantages of extremely polymorphism, high stability, high specificity, co-dominant inheritance, fast detection, publicly ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12N15/12C12Q1/68C12Q3/00
Inventor 胡景杰包振民汪小龙战爱斌惠敏陆维
Owner OCEAN UNIV OF CHINA
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