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A method for the enrichment, extraction and identification of the mitochondrial genome of the river clam

A mitochondrial genome and identification method technology, applied in the field of molecular biology, can solve problems such as difficult to achieve, small total amount, low extraction purity, etc., and achieve the effect of large total amount, good discrimination, and good DNA integrity

Active Publication Date: 2021-09-17
FRESHWATER FISHERIES RES INSITUTE OF JIANGSUPROVINCE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to overcome the defects of low purity and small amount of mitochondrial genome extraction in the prior art, and it is difficult to meet the requirements of follow-up experiments, and to provide a method for enrichment, extraction and identification of mitochondrial genomes. Through the amplification of mitochondrial gene fragments, the Identification of species for molecular biology

Method used

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  • A method for the enrichment, extraction and identification of the mitochondrial genome of the river clam
  • A method for the enrichment, extraction and identification of the mitochondrial genome of the river clam
  • A method for the enrichment, extraction and identification of the mitochondrial genome of the river clam

Examples

Experimental program
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Effect test

Embodiment 1

[0045] Example 1 Extraction of Mitochondrial Genomic DNA from River Clams by Differential Centrifugation

[0046] Take 5g of fresh river clam collected in Jiangsu, China, take its muscle and grind it thoroughly in a pre-cooled glass homogenizer, filter the ground sample with a 1um filter membrane, and centrifuge the collected filtrate with a centrifugal force of 3000g 20 minutes. Transfer the centrifuged supernatant to a new centrifuge tube, centrifuge at 15,000g for 20 minutes, collect the precipitate, add 1ml suspension buffer to the precipitate, and gently suspend the precipitate with a brush. Centrifuge the suspension at 3000g for 10 minutes, take the supernatant, add 2ug DNase I per gram of fresh weight, digest at 35°C for 2 hours, add 0.5mol / L EDTA to stop the digestion reaction, centrifuge at 18000g for 20 minutes, collect the precipitate and get the crude Lift the mitochondria.

[0047] Add 1 / 10 volume of 10% SDS and 1 / 100 volume of 30mg / ml RNase to the crudely extra...

Embodiment 2

[0062] Embodiment 2 primer pair Mit1 amplification and identification

[0063] This study is divided into river clam group, Cyprinidae: silver globber group, Bayesian group, wheat ear fish group, similar bream group, triangular sail mussel group and patina group.

[0064] The operation method of mitochondrial genomic DNA enrichment and extraction of samples to be identified is the same as that in Example 1.

[0065] Using the mitochondrial genomic DNA of each sample to be identified as a template, the primer pair Mit1:Mit1F:5'-ATGGTTTAACGGCTGCGATTGAA-3'(SEQ ID No.3) and Mit1R:5'- CCAACATCGAGGTAGCAAACTTTCT-3' (SEQ ID No. 4) was PCR amplified.

[0066] Before performing PCR amplification, the reagents used were put on ice for thawing, the concentrations of the primers were diluted to 5umol / L, and the total PCR reaction system was 20ul. The reaction system is as follows: 5×FastPfu Buffer 4ul, 2ul 2.5 mMdNTPs, Forward Primer (5μM) and Reverse Primer (5μM) 0.8ul each, FastPfu P...

Embodiment 3

[0074] Embodiment 3 primer pair Mit2 amplification and identification

[0075] This study is divided into river clam group, Cyprinidae: silver globber group, Bayesian group, wheat ear fish group, similar bream group, triangular sail mussel group and patina group.

[0076] The operation method of mitochondrial genomic DNA enrichment and extraction of samples to be identified is the same as that in Example 1.

[0077] Using the mitochondrial genomic DNA of each sample to be identified as a template, the primer pair Mit2: Mit2F:5'-ATCTTACGGGTACTACGAACAAACG-3'(SEQ ID No.5) and Mit2R:5'-ACACCTCTACGGACGCCTCT- 3' (SEQ ID No.6) was amplified by PCR.

[0078] Before performing PCR amplification, the reagents used were put on ice for thawing, the concentrations of the primers were diluted to 5umol / L, and the total PCR reaction system was 20ul. The reaction system is as follows: 5×FastPfu Buffer 4ul, 2ul 2.5 mMdNTPs, Forward Primer (5μM) and Reverse Primer (5μM) 0.8ul each, FastPfu P...

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Abstract

The invention provides a method for enriching and extracting the mitochondrial genome DNA of the clam and identifying the clam through the mitochondrial gene. Through the enrichment and extraction method provided by the present invention, a sufficient amount of mitochondrial genome DNA of the river clam can be successfully obtained. The present invention designs the primer pair Mit1 of the nad2-cox3 gene according to the mitochondrial sequence of the clam, and the primer pair Mit2 designed according to the cob gene fragment. The two pairs of primers provided by the present invention have strong specificity and can specifically amplify the mitochondrial sequence of the clam , but the gene fragment cannot be amplified for the mitochondria of other species. It has the advantages of good amplification performance, good discrimination, and less by-products. It can be used for species identification of river clams.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and relates to a method for enriching, extracting and identifying the mitochondrial genome of the clam, in particular to specifically identifying the mitochondria of the clam through a pair of specific primers. Background technique [0002] River clams (Corbicula sp.), also known as clams, sand snails, snails, etc., belong to the clambranchia, euplobranchial order, clam family, clam genus. River clam is a bivalve aquatic organism with strong environmental adaptability, widely distributed in China, Korea, Japan and Southeast Asian countries. In my country's inland waters, river clams live in caves on the surface of the underwater soil and feed on plankton. They grow fast and have strong reproductive capabilities, making them suitable for artificial breeding. River clam meat is rich in nutrients and can be eaten fresh or used as medicine. It has the functions of appetizing, improving eye...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/686C12Q1/6888C12Q1/6806
CPCC12Q1/6806C12Q1/686C12Q1/6888C12Q2535/122C12Q2565/113C12Q2565/125C12Q2521/327C12Q2521/301
Inventor 张彤晴殷稼雯唐晟凯李大命刘小维刘燕山谷先坤
Owner FRESHWATER FISHERIES RES INSITUTE OF JIANGSUPROVINCE
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