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Method for rapidly detecting microsatellite markers of Charybdis feriatus

A technology of microsatellite marking and detection method, which is applied in the direction of determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of limiting the development of C. rust, no micro-satellite marking of C. rust, and achieving a clear amplification band. Effect

Inactive Publication Date: 2013-09-18
EAST CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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Problems solved by technology

Microsatellite markers are ideal markers for carrying out research in this area, but there are no microsatellite markers available for rusty spot, which limits the development of genetic research on rusty spot

Method used

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  • Method for rapidly detecting microsatellite markers of Charybdis feriatus
  • Method for rapidly detecting microsatellite markers of Charybdis feriatus
  • Method for rapidly detecting microsatellite markers of Charybdis feriatus

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Embodiment 1

[0034] 1. Extraction of Genomic DNA

[0035] Cut the muscle tissue (100-150mg) of Rust spotted worm and place it in 500μl tissue extract solution (10mmol / L Tris-Cl, pH8.0; 100mmol / L EDTA, pH8.0; 100mmol / L NaCl; 0.5%SDS) Cut into pieces, add proteinase K (final concentration 20mg / ml) and RNase A (final concentration 100μg / ml), mix thoroughly, digest at 55°C for 2-3 hours until clear; mix with phenol, chloroform and isoamyl alcohol solution (volume ratio 25:24:1) twice; then precipitate DNA with 2 volumes of absolute ethanol, wash with 70% ethanol and dry naturally, then dissolve in 50 μl TE buffer (10mmol / L Tris- HCl, pH8.0; 10mmol / LEDTA, pH8.0); finally the DNA concentration was diluted to 100ng / μl, and stored at -20°C for later use;

[0036] 2. Functional gene sequence search and microsatellite primer design

[0037] Search and download the functional gene sequence of Rust spotted cat from the GenBank database, and use the biological software SSRHUNTER1.3 to search for micr...

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Abstract

The invention relates to a method for rapidly detecting microsatellite markers of Charybdis feriatus. The method comprises the following steps of: (1) extracting a genomic DNA (Deoxyribose Nucleic Acid) of the Charybdis feriatus; (2) obtaining a gene sequence containing microsatellite repeats according to the functional gene sequence of the Charybdis feriatus in a GeneBank database; (3) designing a microsatellite marker primer; (4) implementing PCR (Polymerase Chain Reaction) amplification on the genomic DNA of different individuals of the Charybdis feriatus; and (5) detecting denaturing polyacrylamide gel electrophoresis of PCR products. The method has the advantages of rapidness, accuracy, sensitivity and the like, and can intuitively detect the genotype of different individuals of the Charybdis feriatus, so that a polymorphism map of genetic variation of the Charybdis feriatus on a functional gene microsatellite site is obtained quickly; the microsatellite markers can be applied to the fields of analysis on genetic variation and population genetic diversity of the Charybdis feriatus.

Description

technical field [0001] The invention belongs to the field of detection of DNA molecular genetic markers of the rust-spotted catfish, in particular to a rapid detection method of the rust-spotted catfish microsatellite marker. Background technique [0002] Microsatellite DNA (Microsatellite DNA) exists in almost all eukaryotic genomes. It is a simple sequence repeat (Simple Sequence Repeats, SSR) composed of 1-6 nucleotides. It is a new type of molecule developed in the past ten years. Marking technology. Microsatellites have the advantages of large number, random distribution, high polymorphism, strong repeatability, co-dominant inheritance, and simple operation, and are widely used in population genetic diversity analysis, germplasm resource protection and management, and genetic linkage maps. Construction and QTL mapping and other fields. [0003] Scholars at home and abroad have developed microsatellite markers in most important aquaculture animals. For example: GUO et...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 马洪雨夏连军邹雄马凌波马春艳陆建学蒋伟李淑娟刘月星
Owner EAST CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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