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

Amplification primer for amplifying complete sequence of mitochondrial genome of cyprinid fish and amplification method

A mitochondrial genome and mitochondrial technology, applied in the field of molecular biology, can solve problems such as low probability of success, high requirements for sample manipulation, and only for a single

Active Publication Date: 2018-10-02
SOUTH CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
View PDF2 Cites 7 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The first is to design and screen specific primers to amplify the mitochondrial genome of a specific species based on the published mitochondrial genome sequence of species with close relatives. The amplification primer and its application (application number CN102776183A) applied 16 pairs of specific primers to amplify the whole mitochondrial genome sequence of large yellow croaker. When it is necessary to obtain the whole mitochondrial genome sequence of various fishes, it is necessary to design primers according to their close relatives, which is time-consuming and labor-intensive.
The second is to obtain the complete mitochondrial sequence based on high-throughput sequencing technology. The premise of this method is to obtain purified fish mitochondria. This step is difficult and requires high requirements for samples, reagents and operations, and the probability of success is low; The cost of high-throughput sequencing is high, and it is not suitable for the determination of mitochondrial genome sequences of multiple species; in addition, because the principle of high-throughput sequencing technology is to break the DNA and then add adapters and then splice it, it often happens that the obtained sequences cannot be spliced ​​into complete circular mtDNA

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
  • Amplification primer for amplifying complete sequence of mitochondrial genome of cyprinid fish and amplification method
  • Amplification primer for amplifying complete sequence of mitochondrial genome of cyprinid fish and amplification method
  • Amplification primer for amplifying complete sequence of mitochondrial genome of cyprinid fish and amplification method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] Design method of amplification primers for whole mitochondrial genome sequence of Cyprinidae

[0092] 1. Screening of primer design templates

[0093] Log on to the NCBI (http: / / www.ncbi.nlm.nih.gov / ) website and search for Cyprinidae fishes with complete mitochondrial genome sequences. The search finds 382 species of Cyprinidae fishes belonging to 134 genera, and each genus selects For one species, the complete mitochondrial genome sequences of 134 species were downloaded and used as templates for primer design.

[0094] 2. Design primers according to the template sequence

[0095] Using MegAlign software to compare the complete mitochondrial genome sequences of 134 species screened to find relatively conserved regions, the complete mitochondrial genome sequence can be divided into three overlapping long fragments with a length of 5000-7000 bp (fragment 1 , Fragment 2 and Fragment 3), then utilize PrimerPremiers 5.0 software to design nested PCR primers for amplifyin...

Embodiment 2

[0105] The method for amplifying the whole mitochondrial genome of Cyprinidae fishes provided in this embodiment comprises the following steps:

[0106] (1) Extraction of whole-genome DNA of 12 species of carps

[0107] 1.1 Collect 1 wild Hainan squid, red-eyed trout, crucian carp, southern squid, southern white turtle, horse mouth fish, yellow-tailed pomfret, silver carp, silver squid, dace, colorful stone squid, and schizothorax, and take the fish Body back muscles were quickly frozen in liquid nitrogen, and then transferred to a -80°C freezer for genomic DNA extraction.

[0108] 1.2 Use TIANamp Genomic DNA Kit to extract muscle genomic DNA, use a nucleic acid and protein analyzer to measure the concentration and purity of DNA samples, and take 3 μL of DNA for electrophoresis detection on 1% agarose gel. -20°C.

[0109] 2. Amplification of full-length mitochondrial sequences

[0110] 2.1 Primer design and PCR amplification

[0111] See Example 1 for the amplification pri...

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 discloses an amplification primer for amplifying a complete sequence of a mitochondrial genome of cyprinid fish. The amplification primer comprises three groups of outer primers and inner primers respectively used for amplifying three long-fragment sequences of the mitochondrial genome and a pair of sequencing primers. The complete sequence of the mitochondrial genome of the cyprinidfish is obtained by using the amplification primer, and a material foundation is provided for conservation genetics and evolutionary genetics of the cyprinid fish as well as the molecular identification of species. The invention further discloses an amplification method for amplifying the complete sequence of the mitochondrial genome of the cyprinid fish. The method is simple, convenient, efficient, fast, and low in time, labor and money consumption.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a primer and an amplification method for the complete sequence of the mitochondrial genome of carps. Background technique [0002] Mitochondrial DNA (mtDNA) is the genetic material in mitochondria, which is a relatively independent genome in cells. Compared with nuclear DNA, mtDNA has a smaller molecule, simpler structure, maternal inheritance, and a higher mutation rate. DNA polymorphism sites formed after mutation fixation can reflect population genetic characteristics, population differentiation, and species relationship etc. Like the mtRNA of other vertebrates, fish mtDNA is covalently closed circular, and it is a genetic factor with autonomous replication, transcription and translation capabilities outside the nucleus. Fish mtDNA mainly includes 37 coding genes (13 hydrophobin genes, 2 rRNA genes, 22 tRNA coding genes), a control region (D-loop) respo...

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): C12Q1/6888C12Q1/6806C12N15/11
CPCC12Q1/6806C12Q1/6888C12Q2531/113C12Q2565/125C12Q2549/119
Inventor 王鹏飞邱丽华赵超范嗣刚闫路路
Owner SOUTH CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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