Detection method for Apostichopus japonicas AjE101 micro-satellite DNA label

A detection method, the technology of aje101, is applied in the direction of microbial determination/inspection, biochemical equipment and methods, etc., to achieve the effect of simple method

Inactive Publication Date: 2011-08-03
SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are few studies on EST microsatellite markers of A. japonicus

Method used

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  • Detection method for Apostichopus japonicas AjE101 micro-satellite DNA label
  • Detection method for Apostichopus japonicas AjE101 micro-satellite DNA label
  • Detection method for Apostichopus japonicas AjE101 micro-satellite DNA label

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Experimental program
Comparison scheme
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Embodiment 1

[0020] 1. Extract DNA from Apostichopus japonicus

[0021] The japonicus japonicus material used in this experiment was randomly extracted from 24 DNA samples, and DNA was extracted from frozen muscle tissue. Cut 100mg of muscle tissue, mince it and place it in 0.7 ml of extraction buffer (6 M Urea (urea), 10 mM Tris-HCl, 125 mM NaCl (sodium chloride), 1% SDS (dodecyl muscle tissue) sodium sulfate), 10 mM EDTA (ethylenediaminetetraacetic acid), pH=7.5), add proteinase K (20 mg / ml) at a final concentration of 0.1 mg / ml, and digest overnight at 37°C. The reactant was extracted three times with phenol:chloroform (1:1), and the extracted supernatant was extracted once with an equal volume of chloroform. The supernatant was precipitated with isopropanol, and then dissolved in 500 1μl×TE (10 mM Tris.HCl, 1 mM EDTA, pH=8.0). Treat the DNA sample with RNase (20 μg / m1) at 37°C for 1 hour, and then use phenol / chloroform extraction solution for DNA purification. Extract once with pheno...

Embodiment 2

[0028] Screening of Microsatellite Loci and Determination of Polymorphic Markers

[0029] 1. Source of microsatellite loci and screening of microsatellite sequences

[0030]Collect and download (in FASTA format) existing EST sequences from the NCBI (http: / / www.ncbi.nlm.nih.gov) database, use SSRhunter software to search and analyze the 5,736 sequences obtained one by one, for 2-6 bases Microsatellite DNA sequences with basic repeating units and repeats greater than 5 were isolated. The ESTs containing microsatellites were clustered and analyzed using SeqMan in the software DNAstar, and the ESTs clustered in different contigs were selected to design primers.

[0031] 2. Design of Microsatellite Marker Primers

[0032] Use primer design software Primer Premier 5.0 and Primerselect in DNAstar to design primers on the flanking sequences of microsatellite repeats; primers are required to meet the following conditions: (1) The length of the primer is 17-25bp; (2) The GC content is...

Embodiment 3

[0041] Screening of Microsatellite Loci and Determination of Polymorphic Markers

[0042] 1 Source of microsatellite loci and screening of microsatellite sequences

[0043] Collect and download (in FASTA format) existing EST sequences from the NCBI (http: / / www.ncbi.nlm.nih.gov) database, use SSRhunter software to search and analyze the 5,736 sequences obtained one by one, for 2-6 bases Microsatellite DNA sequences with basic repeating units and repeats greater than 5 were isolated. The ESTs containing microsatellites were clustered and analyzed using SeqMan in the software DNAstar, and the ESTs clustered in different contigs were selected to design primers.

[0044] 2 Design of microsatellite marker primers

[0045] Use primer design software Primer Premier 5.0 and Primerselect in DNAstar to design primers on the flanking sequences of microsatellite repeats; primers are required to meet the following conditions: (1) The length of the primer is 17-25bp; (2) The GC content is ...

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Abstract

The invention belongs to the field of molecular biology DNA labeling technology and application, and in particular relates to Apostichopus japonicas express sequence tag EST micro-satellite label screening development and application. The invention provides Apostichopus japonicas AjE101 micro-satellite specific DNA primers, a polymerase chain reaction (PCR) reaction system by utilizing the primers, and a detection method for an Apostichopus japonicas AjE101 micro-satellite DNA label. The detection method comprises the following steps of: extracting genome of Litopenaeus vannamei Boone; designing the specific primers at two ends of an Apostichopus japonicas AjE101 micro-satellite DNA core sequence; and performing PCR amplification on genome DNA of different groups of the Litopenaeus vannamei Boone or individuals in the group by using the primers, analyzing products, and determining the genome of each individual so as to obtain an Apostichopus japonicas polymorphism genetic variation map. The invention is mainly applied to Apostichopus japonicas germ plasm resource and genetic diversity analysis, cular population genetics, construction of genetic maps and research of functional genes.

Description

technical field [0001] The invention belongs to the field of molecular biology DNA labeling technology and application, in particular to sea cucumber imitation japonicus (Apostichopus japonicus ) EST (Express Sequence Tag, expressed sequence tag) microsatellite marker screening development and application. Background technique [0002] Apostichopus japonicus is an animal of the genus Apostichopus japonicus in the family Apostichopaceae, commonly known as japonicus japonicus. Distributed in the North Pacific region, including Russia, Japan, the coast of the Korean Peninsula, and China, it is common in harbors with calm waves and currents, lush seagrass, and no freshwater injection. The bottom is rocky or hard bottom. Sea cucumber imitation is the best quality, the most widely distributed and the largest yield among edible sea cucumbers. In my country, the sea cucumber aquaculture industry is mainly distributed in coastal areas such as Liaoning, Hebei, Shandong, Jiangsu and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 王艳红胡超群任春华夏建军
Owner SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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