Next generation sequencing platform-based noninvasive target mitochondrion sequencing method

Abstract

The invention discloses a next generation sequencing platform-based noninvasive target mitochondrion sequencing method. The method comprises the following steps of preparing a sample slice and performing staining; capturing mitochondria through laser microdissection, wherein the number of mitochondrion tissues captured through the laser microdissection is 1-200; performing DNA extraction; performing PCR amplification and product detection; performing library construction and quality inspection; and performing computer sequencing and result analysis. Through a specific staining technology, the mitochondrion tissues are stained; by utilizing a laser microdissection technology, mitochondrial DNAs are obtained in a targeted way; through micro DNA extraction and amplification technologies, the mitochondrial DNAs are obtained and used for downstream library construction and detection; and the experimental design is rigorous, the experimental process is simple, a detection result is accurate and reliable, the scientific research and clinical detection demands can be met to varying degrees, the method is suitable for quick detection of mitochondrion-related mutation in scientific research and clinic, and correct diagnosis and classification of clinical diseases are facilitated.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a non-invasive targeted mitochondrial sequencing method based on a second-generation sequencing platform. Background technique [0002] The mitochondrial genome (mitochondrial DNA, mtDNA) is a closed double-stranded circular structure with a length of 16569bp, which is divided into coding regions and non-coding regions. There are 37 genes in the coding region, of which 22 encode transfer ribonucleic acid (tRNA), 2 encode ribosomal ribonucleic acid (12S and 16SrRNA), and 13 encode polypeptides. The mtDNA genes are closely arranged, and there are no introns between the sequences. [0003] 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...

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Problems solved by technology

[0003] 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

[0004] 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; in the detection of mitochondrial diseases, the most difficult problem to overcome is the high false positive test results. The main reason is the interference of the genome, which leads to the inability to automatically analyze the results, which brings difficulties to the clinical diagnosis of mitochondrial diseases

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