Method for quantitatively detecting nuclear genome copy number and human mitochondrial genome copy number by fluorescence

Abstract

The invention discloses a method for quantitatively detecting nuclear genome copy number and human mitochondrial genome copy number by fluorescence. The method includes respectively selecting a singlecopy gene in human nuclear genome DNA and mitochondrial genome, designing primers and probes, transferring sequence fragments into PMD-18T vectors, extracting plasmids after conversion to obtain thecopy number and taking the copy number as a reference standard; and in actual detection, taking the Cq value of the reference standard to make a standard curve to compare and obtain the copy number ofnuclear genome DNA and mitochondrial genome DNA in a sample to be detected. The method provides a method for quantitatively analyzing the copy number of the mitochondrial genome in different human tissue cells, has high specificity and simple operation, and can complete rapid detection of the copy number of the mitochondrial genome only by a fluorescence quantitative PCR instrument.

Description

technical field [0001] The invention relates to a technique in the field of molecular biology, in particular to a method for quantitatively detecting the copy number of nuclear genome and the copy number of human mitochondrial genome based on TERT and 16S_rRNA gene fluorescence quantitative detection. Background technique [0002] With the rapid development of high-throughput sequencing technology and the improvement of bioinformatics technology, full sequencing analysis of mtDNA has gradually become the main method for mtDNA detection. It can enable forensic workers to obtain more comprehensive mitochondrial genomic DNA information, improve the recognition rate of mtDNA detection, and better analyze the heterogeneity of mtDNA. In the application of forensic evidence, it is necessary to accurately quantify the initial copy number of mtDNA to ensure the accuracy of the analysis results. [0003] How to accurately determine the copy number of mtDNA is a difficult problem. On ...

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Problems solved by technology

[0003] How to accurately determine the copy number of mtDNA is a difficult problem. On the one hand, the mitochondrial organelle is very small, and its genomic DNA is only 16569bp. At the same time, there can be multiple sets of mitochondrial genomic DNA in a cell, so it is difficult to use physical and chemical methods such as electron microscopy or staining. On the other hand, when extracting mtDNA, the product often contains nDNA, and direct detection methods such as spectrophotometer (NanoDrop) or Qubit cannot accurately reflect the copy number of mtDNA

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