Primers and amplification method for complete sequence amplification of mitochondrial genome of Cyprinidae

Abstract

The invention discloses a primer for amplifying the whole genome sequence of carps, including three sets of outer primers and inner primers respectively used for amplifying three long-segment sequences of the mitochondrial genome, and a pair of sequencing primers. The whole mitochondrial genome sequence of carps can be obtained by using the amplification primers, which provides a material basis for conservation genetics, evolutionary genetics and species molecular identification of carps. The invention also discloses a method for amplifying the whole mitochondrial genome of carps. The method is simple, efficient and fast, and saves time, labor and money.

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...

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

Images