Human mitochondria whole genome high-throughput sequencing method based on multiple PCR

Abstract

The invention provides a primer set used for human mitochondria whole genome sequencing and a sequencing method using the primer set. The invention further provides an amplification system which comprises the primmer set and a kit. According to the primer set used for human mitochondria whole genome sequencing and the sequencing method using the primer set, the sequencing method is simple and fast, requirements on the qualities of the samples are not high, sequencing results are accurate and reliable, and deep sequencing on the mitochondria whole genome can be achieved; in addition, the primer set can be used for detecting low frequency mutation in the mitochondria.

Description

technical field [0001] The invention relates to the field of biological detection, in particular to a high-throughput sequencing method for the whole genome of human mitochondria. Background technique [0002] Mitochondria are important organelles in eukaryotic cells and play a vital role in some biological processes, such as oxidative stress, metabolism, inflammation, autophagy, and apoptosis. Although most proteins in mitochondria are encoded by nuclear DNA, proteins encoded by mitochondrial DNA (mtDNA) also play crucial roles. [0003] In each human cell, there are 1,000 to 10,000 mitochondria; in each mitochondria there are 2-10 copies of mitochondrial DNA (mtDNA). Human mitochondrial DNA is a double-stranded closed circular DNA with a full length of 16569bp. It contains coding region and non-coding region, in which the coding region encodes 13 proteins, 22 tRNAs and 2 rRNAs, and the non-coding region has a replication initiation region and some transcriptional regulat...

AI Technical Summary

Problems solved by technology

The traditional method for enriching mitochondrial DNA is the cesium chloride density gradient centrifugation method, which can obtain relatively pure mitochondrial DNA, but this method is time-consuming and laborious, requires a very large sample size, and has high requirements for samples

There are other methods such as hybridization capture, long fragment PCR, hybridization capture enrichment of mitochondrial DNA is expensive and cumbersome to operate, while long fragment PCR method is simple to operate, but requires relatively high sample integrity

At the same time, there are also some methods for mitochondrial sequencing using multiplex PCR, but the existing methods cannot guarantee full mitochondrial coverage at a relatively high and uniform sequencing depth.

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