Microsatellite fluorescent multiple PCR (Polymerase Chain Reaction) method used in paternity testing of grass carps

A paternity test, microsatellite technology, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of larvae survival impact, larvae cannot be marked, etc., to improve accuracy and overcome allele size interpretation errors , the effect of improving efficiency

Active Publication Date: 2014-04-30
YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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Problems solved by technology

Physical marking can be used for individual identification, but this method has great limitations for juvenile fish, eithe

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  • Microsatellite fluorescent multiple PCR (Polymerase Chain Reaction) method used in paternity testing of grass carps
  • Microsatellite fluorescent multiple PCR (Polymerase Chain Reaction) method used in paternity testing of grass carps
  • Microsatellite fluorescent multiple PCR (Polymerase Chain Reaction) method used in paternity testing of grass carps

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Embodiment Construction

[0036] The specific implementation of the present invention will be further described below in conjunction with the examples.

[0037] 1. Grass carp sample DNA extraction

[0038] A total of 8 parents and 94 corresponding offspring were collected from 4 grass carp families. The fin rays were cut and preserved, and the genomic DNA was extracted by the high-salt method: put a small amount of fin rays into an EP tube, add 500 μL HOM Buffer (80 mmol / L EDTA , 100mmol / L Tris, 0.5% SDS, pH8.0) and 10μL of proteinase K at a concentration of 20mg / mL, digested at 55°C for 3-12 hours until the solution was completely transparent; Chloroform, mix well for 15min, centrifuge at 10000r / min for 10min; transfer the supernatant to another centrifuge tube; add 600μL of isopropanol, mix well, centrifuge at 13000r / min for 10min, discard the supernatant; add 0.5mL volume percentage Wash with 70% ethanol for 5 minutes, centrifuge at 13000r / min for 10 minutes, discard the supernatant; Or dissolved ...

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Abstract

The invention discloses a microsatellite fluorescent multiple PCR (Polymerase Chain Reaction) method used in paternity testing of grass carps. The method comprises the following steps: 1, extracting DNA (Deoxyribonucleic Acid) of an individual grass carp individual; 2, sieving a grass carp polymorphism microsatellite primer; 3, optimizing and amplifying a grass carp microsatellite multiple PCR condition; 4, performing paternity testing. According to the method, microsatellite marking and multiple fluorescent PCR technologies are combined, thirteen high-polymorphism microsatellite sites are sieved, three multiple fluorescent PCR systems are constructed, and high-throughput individual identification and parenthood analysis are performed on grass carp families through typing of a sequencer. By adopting the method, a novel technical measure is provided for the germplasm identification, family management and evaluation of multiplication release effect of grass carps.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a method for microsatellite fluorescent multiple PCR for paternity identification of grass carp. Background technique [0002] Grass carp is one of the traditional "four major fishes" in China. Its origin is in the Yangtze River, Pearl River, Heilongjiang and other water systems. It has the advantages of fast growth, easy feeding, and high nutritional value. It has been widely introduced and cultivated in my country and all over the world. One of the main freshwater cultured species. In recent decades, due to factors such as environmental pollution, overfishing, and construction of water conservancy facilities, the natural resources of grass carp in rivers such as the Yangtze River have been decreasing. In order to protect the germplasm resources of the four major Chinese carps, my country has carried out research on the germplasm resources of the four major domestic carps since the...

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q1/6888C12Q2600/156C12Q2537/143
Inventor 汪登强蒋菁菁段辛斌刘绍平陈大庆刘明典王珂李树华
Owner YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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