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A Sanger-based method for sequencing the human mitochondrial genome

A technology of human mitochondria and genome, applied in the field of genetic engineering, can solve problems such as cumbersome operation, nuclear DNA pollution, increased sequencing workload, etc., and achieve the effect of avoiding the experimental operation process and reducing the amount of data

Active Publication Date: 2019-02-12
SHANGHAI PASSION BIOTECHNOLOGY CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, to obtain mtDNA with higher purity, the operation is cumbersome, and the experimental process is a little careless. The obtained mtDNA will be contaminated by nuclear DNA, and a large amount of raw data will greatly increase the workload of sequencing.

Method used

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  • A Sanger-based method for sequencing the human mitochondrial genome
  • A Sanger-based method for sequencing the human mitochondrial genome
  • A Sanger-based method for sequencing the human mitochondrial genome

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Embodiment

[0026] This embodiment relates to a method for sequencing the human mitochondrial genome based on Sanger; specifically, it includes the following steps:

[0027] 1. Genome extraction: extraction of human total DNA

[0028] 1. Take 1-20 mg of animal tissue, transfer it into a mortar pre-cooled in an ice-water bath, and quickly and vigorously grind it into a homogenate.

[0029] 2. After adding 350 μl Buffer PBS and 0.9 μl RNase A, grind gently for 30 seconds.

[0030] 3. Collect 350 μl of ground tissue homogenate and transfer to a 2ml centrifuge tube. If the homogenate volume is less than 350 μl, add PBS to 350 μl.

[0031] 4. Add 150 μl Buffer C-L and 20 μl Proteinase K. Immediately vortex for 1 min to mix well. After brief centrifugation, place the centrifuge tube in a 56°C water bath for 10 minutes.

[0032] *Do not add Proteinase K directly to Buffer C-L.

[0033] 5. Add 350μl Buffer P-D, vortex for 30s to mix well, and centrifuge at 12,000×g for 10min.

[0034] 6. P...

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Abstract

The invention discloses a method for sequencing human mitochondrial genome based on Sanger. The method comprises the following steps: extracting human total DNA; designing a pair of primers covering the full length of mitochondria according to the sequence of human mitochondria measured in the NCBI database. DNA is amplified by PCR; PCR amplification products are recovered to obtain human mitochondria-specific gene fragments; the gene fragments are sequenced; the measured sequences are spliced ​​by CExpress according to the sequence overlap between the primer pair and the primer pair, The complete sequence of human mitochondria was assembled. The present invention only needs to extract human total DNA, avoiding the tedious experimental operation process of extracting relatively high-purity mtDNA, and then according to the human mitochondrial sequence measured in the NCBI database, a primer pair covering the full length of mitochondria is designed to amplify the total DNA, Obtain mitochondria-specific gene fragments for sequencing, reducing the amount of data for total DNA sequencing.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for sequencing human mitochondrial genome based on sanger. Background technique [0002] Since the discovery of mitochondria, a large amount of information has been accumulated in terms of its morphological structure, gene composition, replication, transcription and translation, and the relationship with the nuclear genome, as well as its genetic characteristics, evolutionary characteristics and molecular systematics. Mitochondria are important organelles in eukaryotic cells, where energy is generated, and are also involved in the synthesis of fatty acids and certain proteins. Mitochondrial DNA is a relatively independent genome in cells. Mitochondrial DNA is a small and easy-to-purify replication transcription unit in cells. The genome structure is relatively simple, and has high specificity and uniqueness. Its transmission, recombination, separation, replication, an...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6869
CPCC12Q1/6869
Inventor 孙子奎王锋丁方美李嫦娥方婉
Owner SHANGHAI PASSION BIOTECHNOLOGY CO LTD
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