Free mitochondrial DNA mutation detection technique based on bibasic sequencing technique

A second-generation sequencing technology and mutation detection technology, which is applied in the field of free mitochondrial DNA mutation detection technology, can solve the problems of false positives and low mutation frequencies in low-frequency mutation detection, and achieve the goal of improving the comparison rate, improving accuracy, and overcoming false positives Effect

Pending Publication Date: 2019-10-08
FOURTH MILITARY MEDICAL UNIVERSITY
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Problems solved by technology

[0004] The technical problem to be solved in the present invention is to overcome the above technical defects and provide a free mitochondrial DNA mutation detection technology based on next-generation sequencing technology, aiming at the characteristics of high degree of fragmentation and low mutation frequency of free mtDNA, enriching free mtDNA in NGS Sequence information, to solve the false positive problem of its low-frequency mutation detection, overcome the interference of NUMT for mutation detection, and realize the accurate detection of free mitochondrial DNA mutation

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  • Free mitochondrial DNA mutation detection technique based on bibasic sequencing technique
  • Free mitochondrial DNA mutation detection technique based on bibasic sequencing technique
  • Free mitochondrial DNA mutation detection technique based on bibasic sequencing technique

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Embodiment Construction

[0015] The present invention will be described in further detail below in conjunction with the accompanying drawings.

[0016] A free mitochondrial DNA mutation detection technology based on next-generation sequencing technology, mainly comprising the following steps:

[0017] 1) Preparation of cfDNA single-strand sequencing library: dephosphorylate the end of the extracted cfDNA fragment and denature it into a single strand; use DNA ligase to connect adapters on both sides of the cfDNA fragments, and the adapters contain 12bp random base sequences; through primer extension , changing the single-stranded DNA library into a double-stranded library, and changing the cfDNA fragment into a single-stranded sequence library construction can enrich fragments shorter than 150bp, which is conducive to increasing the proportion of free mtDNA in the genome-wide library; UMI of 12bp can eliminate False positives due to PCR and sequencing errors.

[0018] 2) mtDNA capture: After explorati...

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Abstract

The invention discloses a free mitochondrial DNA mutation detection technique based on a bibasic sequencing technique. The free mitochondrial DNA mutation detection technique mainly comprises the following steps of (1) preparing a cfDNA single-chain sequencing library; (2) performing mtDNA capturing; and (3) performing data analysis. Compared with the prior art, the free mitochondrial DNA mutationdetection technique has the advantages that in accordance with the characteristics of high gragmentation degree and low mutation frequency of free mtDNA, free mtDNA sequence information is enriched in NGS, so that the low-frequency mutation detection false positive problem is solved, interference of NUMT to mutation detection is overcome, and pinpoint detection of free mitochondrial DNA mutationis realized.

Description

technical field [0001] The invention relates to the field of biomedical technology, in particular to a free mitochondrial DNA mutation detection technology based on next-generation sequencing technology. Background technique [0002] As the most powerful complement to traditional methods, liquid biopsy technology has made breakthroughs in the clinical application of new markers. Among them, cell-free DNA detection has become the most concerned and fastest-growing liquid biopsy method. Mitochondrial DNA (mtDNA) has a large copy number, and the average copy number of mtDNA in each cell can reach 103; the genome is small, the full length of human mtDNA is only 16,569bp, the amount of data generated by sequencing is small, and the detection is fast and cost-effective; mutation rate High, because mtDNA often exists in the environment of high levels of active oxygen, and lacks histone protection and effective DNA repair system, so its mutation frequency is about 10 times higher t...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6869G16B30/10
CPCC12Q1/6869G16B30/10C12Q2525/191C12Q2565/519
Inventor 邢金良刘洋尹纯
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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