Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Corbicula fluminea mitochondrial genome enrichment, extraction and indentifying method

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

Active Publication Date: 2019-01-01
FRESHWATER FISHERIES RES INSITUTE OF JIANGSUPROVINCE
View PDF1 Cites 2 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Corbicula fluminea mitochondrial genome enrichment, extraction and indentifying method
  • Corbicula fluminea mitochondrial genome enrichment, extraction and indentifying method
  • Corbicula fluminea mitochondrial genome enrichment, extraction and indentifying method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Extraction of Mitochondrial Genomic DNA from River Clams by Differential Centrifugation

[0045] 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.

[0046] Add 1 / 10 volume of 10% SDS and 1 / 100 volume of 30mg / ml RNase to the crudely extra...

Embodiment 2

[0061] Embodiment 2 primer pair Mit1 amplification and identification

[0062] 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.

[0063] The operation method of mitochondrial genomic DNA enrichment and extraction of samples to be identified is the same as that in Example 1.

[0064] 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.

[0065] 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 2.5mMdNTPs, Forward Primer (5μM) and Reverse Primer (5μM) 0.8ul each, FastPfu Po...

Embodiment 3

[0073] Embodiment 3 primer pair Mit2 amplification and identification

[0074] 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.

[0075] The operation method of mitochondrial genomic DNA enrichment and extraction of samples to be identified is the same as that in Example 1.

[0076] 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.

[0077] 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 2.5mMdNTPs, Forward Primer (5μM) and Reverse Primer (5μM) 0.8ul each, FastPfu Poly...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention provides an enrichment and extraction method of mitochondrial whole genome DNA of corbicula fluminea, and a method of identifying the corbicula fluminea through mitochondrial genes. Through the enrichment and extraction method provided by the invention, the sufficient mitochondrial genome DNA of the corbicula fluminea can be successfully obtained. A primer pair Mit1 of an nad2-cox3 gene is designed according to the mitochondrial sequence of the corbicula fluminea, and a primer pair Mit2 is designed according to a cob gene segment; the two primer pairs provided by the invention are high in specificity, the mitochondrial sequence of the corbicula fluminea can be specifically amplified, and for mitochondria of other species, the gene segment cannot be formed by amplification; and the advantages of good amplification performance and discrimination, few by-products, and the like are achieved, and the primer pairs can be used for species identification of the corbicula fluminea.

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com