A method for non-invasive detection of mitochondrial DNA based on high-throughput gene sequencing

Abstract

The invention provides a method for carrying out non-invasive measurement on mitochondrial DNA (deoxyribonucleic acid) based on high-flux gene sequencing. The method comprises the following steps: (1), extracting a total genomic DNA of an oral cell; (2), designing a PCR (Polymerase Chain Reaction) primer and enriching and amplifying the mitochondrial DNA in vitro; and (3), detecting high-flux sequencing. The method provided by the invention has the advantages that experimental design is precise, experimental flow is simple, a detection result is accurate, and the method is rapid, high in automation degree, capable of meeting demands of scientific research and clinical detection from different degrees, and suitable for relevant mutation rapid detection specific to mitochondria of scientific research and clinical application.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a method for non-invasive detection of mitochondrial DNA sequences. Background technique [0002] The human mitochondrial DNA genome (mitochondrial DNA, mtDNA) is a closed double-stranded circular molecule with a full length of 16569bp. The NCBI accession number of the mitochondrial DNA genome is NC_012920. There are 37 genes in the coding region of the whole genome, and 22 of these 37 genes are One encodes transfer ribonucleic acid (tRNA), two encodes ribosomal ribonucleic acid (12S and 16S rRNA), and 13 encode polypeptides. The mtDNA gene was closely arranged, and no introns were found between the sequences. mtDNA has unique maternal inheritance characteristics, and it is generally believed that mtDNA is passed on to offspring along with the mother's egg cells; the copy number of mtDNA in cells is very high, and a cell often contains up to thousands of mtDNA copi...

AI Technical Summary

Problems solved by technology

mtDNA has unique maternal inheritance characteristics, and it is generally believed that mtDNA is passed on to offspring along with the mother's egg cells; the copy number of mtDNA in cells is very high, and a cell often contains up to thousands of mtDNA copies, and mtDNA has a high mutation rate. A large number of studies have shown that the frequency of mtDNA mutations is more than 10 times higher than that of the nuclear genome. Mutated mtDNA and wild-type mtDNA can coexist in a cell to exert different biological functions. Most mtDNA mutations are harmful mutations. Clinical manifestations caused by mtDNA mutations The type is extremely wide, involving almost all tissues and organs. The harm caused by mtDNA mutations has a dose effect. Slight mutations do not appear typical clinical symptoms, which brings great difficulties to clinical diagnosis. Many mitochondrial diseases cannot be diagnosed Therefore, the accurate detection of mitochondrial genome has important clinical guiding significance

[0003] Traditional mitochondrial gene detection methods mainly include: PCR-RFLP, AS-PCR, DNA generation sequencing, chip method, etc. These methods play an important role in the detection of gene mutations, but there are obvious deficiencies in general, and they are often targeted at mtDNA in clinical practice. However, the detection only focuses on the mutation and deletion screening of a few common mitochondrial gene loci, such as the screening of the common mutation A3243G in mitochondria-related diabetes. The positive detection rate is low, and it is difficult for most patients to obtain an accurate etiological diagnosis In addition, mitochondrial diseases have a dose-effect phenomenon, that is, a small amount of mitochondrial DNA mutations may not cause clinical symptoms, but as the proportion of mutant mitochondria increases, clinical manifestations will appear, and the clinical severity may be positively correlated with the mutation proportion. Low-dose mitochondrial mutations cannot be detected well, and many missed detections will occur if traditional methods are used for detection; in addition, traditional detection methods are limited in detecting gene sites, time-consuming, and cumbersome to operate, etc., which are not suitable for large quantities, Systematic detection results in high false positive results, and the results cannot be analyzed automatically, which brings difficulties to the clinical diagnosis of mitochondrial diseases

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