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A mitochondrial genome library based on high-throughput sequencing and its construction method

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

Active Publication Date: 2017-10-13
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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  • A mitochondrial genome library based on high-throughput sequencing and its construction method
  • A mitochondrial genome library based on high-throughput sequencing and its construction method
  • A mitochondrial genome library based on high-throughput sequencing and its construction method

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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

A mitochondrial genomic library based on high-throughput sequencing and a method for constructing the library. The method comprises the following steps: S1. performing a one-step PCR amplification of total mitochondrial DNA from total DNA; S2. fragmenting the total mitochondrial DNA to obtain a DNA fragment; S3. performing end repair on the DNA fragment to obtain a blunt-end DNA fragment; S4. A-tailing a 3' end of the blunt-end DNA fragment to obtain an A-tailed DNA fragment; S5. adding a linker on the 3' end of the A-tailed DNA fragment to obtain a DNA fragment with linkers; S6. performing LM-PCR on the DNA fragment with linkers; and S7. performing PCR on an LM-PCR product. The method for constructing the mitochondrial genomic library performs PCR amplification on total DNA to obtain total mitochondrial DNA, thereby ensuring accurate amplification of a human mitochondrial genome and improving the accuracy and precision of mutation detection by preventing contamination or mixing of a homologous sequence from a human genome.

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 Patents(China)
IPC IPC(8): C12N15/10C12Q1/68C40B50/06C40B40/06
CPCC12N15/10C12Q1/68C40B40/06C40B50/06
Inventor 张巍
Owner GUANGZHOU JIAJIAN MEDICAL TESTING CO LTD
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