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Novel rapid DNA separation and sequencing method for mitochondrial genome and kit

A mitochondrial genome and detection kit technology, applied in the field of DNA detection, can solve the problems of PCR affecting sequencing results, unable to detect, unable to detect mitochondrial sequences, etc.

Pending Publication Date: 2018-04-06
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

2. Use an ultracentrifuge to separate mitochondria and extract DNA for testing; the disadvantage of this is that a large tissue sample is required, and the preliminary experimental process is cumbersome
3. Design primers covering the entire mitochondrial genome, obtain the mitochondrial sequence by PCR, and sequence it; the disadvantage of this is that it cannot detect unknown mitochondrial sequences, the cost of primer design is relatively high, and PCR is easy to introduce mutations that affect the sequencing results. There are always some areas cannot be detected
Neither enzyme has been reported for the enrichment of eukaryotic mitochondrial DNA

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] 1) Take 10 mg fresh sample of Arabidopsis thaliana, use genome kit or liquid nitrogen to grind, remove protein with chloroform, and extract total genomic DNA by isopropanol precipitation, add water to dissolve.

[0030] 2) Take 2ug of total DNA, add 1 μl Plasmid-Safe ATP-Dependent DNase enzyme, 2 μl 10*buffer, 0.8 μl 10 mmol ATP, add water to make up to 20 μl system, 37 degrees, reaction 1- 3 hours.

[0031] 3) Add 30 microliters of ultrapure water to the reaction system, add 40 microliters of AMP XP magnetic beads, mix well, absorb undegraded DNA, wash twice with 70% alcohol, dry for 3 minutes, add 20 microliters liter of ultrapure water to dissolve the DNA.

[0032] 4) Take 100ng of DNA treated with Plasmid-Safe ATP-Dependent DNase enzyme and use Ion PGM TM Template OT2 200Kit constructs a 200bp library, and uses Ion Torrent to personally operate the genome sequencer (PGM TM ) to detect the DNA sequence and obtain 9M data.

[0033] 5) Use the clc genomics workbenc...

Embodiment 2

[0035] 1) Take 10 mg of frozen pork sample, grind it with liquid nitrogen, remove protein with chloroform, and precipitate DNA with isopropanol to extract total genomic DNA, and add water to dissolve it.

[0036] 2) Take 1 ug of total DNA, add 1 microliter of Plasmid-Safe ATP-Dependent DNase enzyme, 2 microliters of 10*buffer, 0.8 microliters of 10 millimolar ATP, add water to make up to 20 microliters of the system, 37 degrees, overnight reaction 12 Hour.

[0037] 3) Add 30 microliters of ultrapure water to the reaction system, add 40 microliters of AMP XP magnetic beads, mix well, absorb undegraded DNA, wash twice with 70% alcohol, dry for 3 minutes, add 20 microliters liter of ultrapure water to dissolve the DNA.

[0038] 4) Utilize Ion PGM TMTemplate OT2 200Kit constructs a 200bp library, and uses Ion Torrent to personally operate the genome sequencer (PGM TM ) to detect the DNA sequence and obtain 17M data.

[0039] 5) Using clc genomics workbench analysis software to...

Embodiment 3

[0041] 1) Centrifuge 10 ml at high speed to take yeast overnight culture solution, discard supernatant, grind with liquid nitrogen, extract total genomic DNA by isopropanol precipitation, add water to dissolve.

[0042] 2) Take 3ug of total DNA, add 3 microliters of Exonuclease III enzyme, 2 microliters of 10*buffer, add water to make up to 20 microliters of the system, and react at 37 degrees for 0.5 hours.

[0043] 3) Add 30 microliters of ultrapure water to the reaction system, add 40 microliters of AMP XP magnetic beads, mix well, absorb undegraded DNA, wash twice with 70% alcohol, dry for 3 minutes, add 20 microliters liter of ultrapure water to dissolve the DNA.

[0044] 4) Take 100 ng of DNA treated with Exonuclease III enzyme, use Ion PGMTTemplate OT2200Kit to construct a 200bp library, and use Ion Torrent Personal Genome Sequencer (PGMTM) to detect the DNA sequence and obtain 7.5M data.

[0045] 5) Using clc genomics workbench analysis software to remove low-quality ...

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PUM

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Abstract

The invention discloses a novel rapid DNA separation and sequencing method for a mitochondrial genome and a kit. A linear chromosome sequence is degraded by Exonuclease III or Plasmid-Safe ATP-Dependent DNase, and then mitochondrial genome DNA is enriched for high-throughput sequencing. The mitochondrial DNA sequencing method is applied to detection of the mitochondrial genome and particularly detection of the mitochondrial genome without a consensus sequence.

Description

technical field [0001] The invention belongs to the technical field of DNA detection, and relates to a new method for rapid separation and sequencing of mitochondrial genome DNA and a kit. Background technique [0002] The vast majority of eukaryotic organisms have mitochondria, which are structures that produce energy in cells and are the main place for cells to perform aerobic respiration, known as biological engines. Mitochondria have their own genetic material and genetic system, but their genomes are small and they are semi-autonomous organelles. In addition to providing energy for cells, mitochondria are also involved in processes such as cell differentiation, cell information transmission and apoptosis, and have the ability to regulate cell growth and cell cycle. Defects in mitochondrial sequences in animals can cause severe disease, and mitochondrial genome sequences are widely used to study species evolution. [0003] Mitochondrial genome sequence is relatively di...

Claims

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Application Information

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IPC IPC(8): C12Q1/6869
CPCC12Q1/6869C12Q2521/319C12Q2535/122
Inventor 黄方亮
Owner ZHEJIANG UNIV
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