Detecting method for Litopenaeus vannamei Boone LvE165 microsatellite DNA marker

A detection method, the technology of Ive165, is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., to achieve the effect of simple method

Inactive Publication Date: 2011-08-10
SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

There are still few studies on EST micro

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  • Detecting method for Litopenaeus vannamei Boone LvE165 microsatellite DNA marker
  • Detecting method for Litopenaeus vannamei Boone LvE165 microsatellite DNA marker
  • Detecting method for Litopenaeus vannamei Boone LvE165 microsatellite DNA marker

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Embodiment 1

[0019] 1. Extraction of DNA from Litopenaeus vannamei

[0020] The materials of Litopenaeus vannamei used in this experiment were randomly extracted 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 ...

Embodiment 2

[0027] Screening of Microsatellite Loci and Determination of Polymorphic Markers

[0028] 1. Source of microsatellite loci and screening of microsatellite sequences

[0029]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.

[0030] 2. Design of Microsatellite Marker Primers

[0031] 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

[0040] 1. DNA extraction of Litopenaeus vannamei

[0041] The 10 Litopenaeus vannamei used in this example were extracted from muscle tissue soaked in 95% ethanol. Wash twice with ddH2O before extraction, and then place in 0.7 ml of extraction buffer (6 M Urea (urea), 10 mM Tris-HCl. 125 mM NaCI (sodium chloride), 1% SDS (dodecyl muscle 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, cut into pieces, 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 phenol / chloroform and once with chl...

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Abstract

The invention belongs to the field of molecular biology DNA marking technology and application, in particular to a detecting method for a Litopenaeus vannamei Boone LvE165 microsatellite DNA marker. The invention provides a Litopenaeus vannamei Boone LvE165 microsatellite specific DNA primer, a PCR (Polymerase Chain Reaction) system utilizing the primer and a detecting method for the Litopenaeus vannamei Boone LvE165 microsatellite DNA marker. The method comprises the following steps of: extracting a Litopenaeus vannamei Boone genome and diluting for later use; designing specific primers at both ends of a sequence by utilizing a Litopenaeus vannamei Boone LvE165 microsatellite DNA core sequence; and then carrying out PCR amplification on genome DNA of different groups of Litopenaeus vannamei Boone or individuals in the group by using the primer, analyzing a product and confirming the genotype of each individual so as to obtain a polymorphism genetic variation map. The invention is mainly applied to Litopenaeus vannamei Boone germplasm resource and genetic diversity analysis, molecular population genetics, construction of the genetic map, and the like.

Description

technical field [0001] The invention belongs to the field of molecular biology DNA marker technology and application, in particular to the marine economic animal Penaeus vannamei (Litopenaeus vannamei) Litopenaeus vannamei ) Detection method of LvE165 microsatellite DNA marker. Background technique [0002] Litopenaeus vannamei ( Litopenaeus vannamei ) is a wide-salt, wide-temperature shrimp. The prawn is native to the Western Hemisphere and is mainly distributed in Central and South America from the southwest coast of Mexico, through Guatemala, El Salvador, Honduras, Nicaragua, Costa Rica, Panama, Colombia, Ecuador to the Pacific coast of western Peru. Litopenaeus vannamei is one of the main cultured prawn species in the world. It is also the most important cultured prawn species in my country at present. Its breeding area is very wide, including not only most of the coastal areas from Liaoning in the north to Hainan in the south, but also the vast inland freshwater aquac...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 夏建军张吕平胡超群王艳红
Owner SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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