Mitochondrial genome library based on high-throughput sequencing and building method thereof

A mitochondrial genome and library construction technology, applied in the field of gene detection, can solve the problems of complicated purification operation and different amount of magnetic beads, and achieve the effect of simplifying operation steps, increasing sequencing depth, and improving mutation detection.

Active Publication Date: 2016-08-31
GUANGZHOU JIAJIAN MEDICAL TESTING CO LTD
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Problems solved by technology

In this invention, the purification of magnetic beads is carried out separately and the amount of magnetic beads is different, and the purification operation is complicated

Method used

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  • Mitochondrial genome library based on high-throughput sequencing and building method thereof
  • Mitochondrial genome library based on high-throughput sequencing and building method thereof
  • Mitochondrial genome library based on high-throughput sequencing and building method thereof

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Embodiment 1

[0058] A high-throughput sequencing-based mitochondrial genome detection and analysis method, comprising the following steps:

[0059] S1, PCR amplification of mitochondrial full-length DNA

[0060] DNA was extracted from 300 μl whole blood according to conventional methods, and 2 μl DNA samples were taken for concentration determination on NanoDrop. The measured DNA concentration requires the OD260 / OD280 ratio to be between 1.8 and 2.0, and the OD260 / OD230 ratio to be between 1.8 and 2.2. The above two ratios can determine the purity of the extracted DNA. If it exceeds the above range, it can be considered that the purity of the extracted DNA does not meet the requirements, and re-extraction or re-purification is required. According to the determined concentration, it was further diluted to a concentration of 50 ng / μl with DNA lysis buffer.

[0061] Using the diluted whole blood DNA as a template, one-step PCR was performed to amplify the full-length mitochondrial DNA. The ...

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Abstract

The invention relates to a mitochondrial genome library based on high-throughput sequencing and a building method thereof. The building method comprises the following steps of S1, performing one-step PCR (polymerase chain reaction) amplification of mitochondria full-length DNA (deoxyribonucleic acid) in total DNA; S2, crushing the mitochondria full-length DNA to obtain DNA fragments; S3, performing tail end restoration on the DNA fragments to obtain flat tail end DNA fragments; S4, adding A to the end 3' of the flat tail end DNA fragments to obtain the A-added DNA fragments; S5, adding a connector to the end 3' of the A-added DNA fragments to obtain connector-added DNA fragments; S6, performing front LM-PCR on the connector-added DNA fragments; S7, performing post PCR on the LM-PCR product. The building method of the mitochondrial genome library has the advantages that the mitochondrial full-length DNA is obtained through further PCR amplification from the total DNA, so that the precise amplification of the human mitochondrial genome can be guaranteed; the pollution mixing by the homologous sequence of the human genome is avoided, so that the mutation detection is more sensitive and accurate.

Description

technical field [0001] The invention relates to the field of gene detection, in particular to a high-throughput sequencing-based mitochondrial genome library and a construction method thereof. Background technique [0002] Mitochondria are the energy-producing organelles within cells. Mitochondrial DNA is the genetic material in the mitochondria, in a double-stranded circular shape. There can be one or several mitochondrial DNA molecules in a mitochondria. Human mitochondrial DNA (mtDNA) contains a total of 37 genes, of which 22 genes encode transfer ribonucleic acid (tRNA), 2 genes encode ribosomal ribonucleic acid (12S and 16SrRNA), and 13 genes encode polypeptides. These genes on the mitochondrial genome are essential for the production of functional proteins by the mitochondria, and the mitochondrial ribosome is a type of ribosome that exists in the mitochondrial matrix and is responsible for the translation work performed in the mitochondria. [0003] Mitochondrial d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/68C40B50/06C40B40/06
CPCC12N15/10C12Q1/68C40B40/06C40B50/06C12N15/1093C12Q1/6806C12Q2531/113C12Q2525/191
Inventor 张巍
Owner GUANGZHOU JIAJIAN MEDICAL TESTING CO LTD
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