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Method for precisely determining high-frequency and low-frequency mutations of mitochondrial DNA (deoxyribonucleic acid) by high-throughput sequencing

A low-frequency mutation and mitochondrial technology, applied in the field of genomics, can solve the problems of poor uniformity of mitochondrial genome coverage, time-consuming and labor-intensive problems

Active Publication Date: 2014-02-26
GRACELL BIOTECH SHANGHAI CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this method can amplify mitochondrial DNA fragments from total DNA, each sample needs to be amplified with more than 10 different primer pairs, and it needs to be purified separately, which is time-consuming and labor-intensive. Efficiencies also vary, and thus less uniform coverage across the mitochondrial genome

Method used

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  • Method for precisely determining high-frequency and low-frequency mutations of mitochondrial DNA (deoxyribonucleic acid) by high-throughput sequencing
  • Method for precisely determining high-frequency and low-frequency mutations of mitochondrial DNA (deoxyribonucleic acid) by high-throughput sequencing
  • Method for precisely determining high-frequency and low-frequency mutations of mitochondrial DNA (deoxyribonucleic acid) by high-throughput sequencing

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Effect test

Embodiment 1

[0022] Embodiment 1: the extraction of total DNA

[0023] Total DNA (including human, orangutan, macaque, rat, mouse, Drosophila, zebrafish, nematode, Arabidopsis, rice, cotton, rapeseed, yeast and Plasmodium) was extracted using Qiagen’s QIAamp DNA Blood Mini kit Reagent test kit. Different cells or tissues were extracted according to the procedures suggested by Qiagen. Typically, 20 mg of tissue is sampled and 200 μl of AL buffer is added. Homogenization was carried out with steel balls in a 2ml round bottom centrifuge tube. The instrument used was a TissueLyser homogenizer from Qiagen Company with a parameter of 50 beats per second for a total of 2 minutes of homogenization. After column purification, elute into 200 μl of pure water. DNA concentration was determined using a Nanodrop spectrophotometer. 100 ng of total DNA was used to construct the mitoRCA-seq library.

Embodiment 2

[0024] Example 2: Mitochondrial DNA Amplification Using Rolling Circle Replication

[0025] 100 ng of total DNA was used for rolling circle amplification. Add 100ng of total DNA to a 50ul reaction system, which also includes: 1x Phi29 DNA polymerase buffer (NEB), 0.2 μg / ml BSA, 1 mM dNTP (NEB) and 25 μM mitochondrial DNA-specific primer (mouse See the sequence listing for primers). The system was mixed and heated at 95°C for 3 minutes, then cooled to room temperature, followed by the addition of 1 μl Phi29 DNA polymerase (10 unit / μl, NEB). After mixing, react at 37°C for 16 hours. This was followed by heating to 65°C for 10 minutes to inactivate the polymerase. Qiagen's REPLI-g Mitochondrial DNA kit can also be used for rolling circle replication.

Embodiment 3

[0026] Example 3: Restriction Enzyme Digestion

[0027] Different species require different restriction enzymes. The principle is to choose 1-2 restriction endonucleases that can make the corresponding mitochondrial DNA produce 2 bands that can be separated on agarose gel electrophoresis. We chose EcoRV (NEB) to generate 2 bands of 9.5kb and 6.8kb. For Drosophila, NdeI (NEB) and EcoRV (NEB) can be used to generate 10kb and 9.8kb bands. For human samples, SacI(NEB) can be used to generate 2 bands at 6.9kb and 9.6kb. The specific restriction enzyme digestion system is implemented according to the recommended dosage of each enzyme.

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Abstract

The invention belongs to the technical field of genomics and specifically relates to a method for precisely determining high-frequency and low-frequency mutations of mitochondrial DNA (deoxyribonucleic acid) by high-throughput sequencing. According to the method provided by the invention, the purpose of determining the high-frequency and low-frequency mutations in a mitochondrial genome is achieved by designing a set of mitochondrial DNA-specific primers, enriching the mitochondrial DNA from total DNA by rolling-circle replication, further enriching the mitochondrial DNA through a specific restriction endonuclease, then constructing a sequencing library, performing high-throughput sequencing and developing a specific data analysis process flow. Mutation sites of which the frequency is as low as 0.3% can be detected to the greatest extent.

Description

technical field [0001] The invention belongs to the technical field of genomics, and specifically relates to a method for detecting high-frequency and low-frequency mutations in eukaryotic mitochondrial DNA by using high-throughput sequencing (next-generation sequencing, second-generation sequencing, and deep sequencing) technology. Background technique [0002] Mitochondria are important organelles that provide energy in cells and play a central role in energy conversion and metabolism in organisms. Mutations or deletions of mitochondrial DNA (deoxyribonucleic acid) in human cells can cause abnormalities in oxidative phosphorylation and energy supply, and in turn lead to the occurrence of nearly 150 human diseases, such as cardiovascular disease, diabetes, deafness, tumors, neurodegeneration Disease and aging etc. [0003] Mitochondrial DNA is extranuclear genetic material, which can independently encode some important proteins involved in mitochondrial function. Human mi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6869C12Q2531/125
Inventor 倪挺朱钧魏刚谌婷
Owner GRACELL BIOTECH SHANGHAI CO LTD
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