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Primer pair and method used for whole genome amplification of human mitochondria

A whole genome amplification and mitochondrial technology, applied in the field of gene amplification, can solve the problems of inability to accurately assist in the diagnosis of etiology, inability to exclude pseudogene interference, deletion of several DNA fragments, etc., to improve accuracy and amplification efficiency, reduce Effects of primer design and optimization time

Inactive Publication Date: 2017-05-31
北京信诺佰世医学检验所有限公司
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Problems solved by technology

[0004] The technology of using multiple pairs of primers to amplify the whole circle of the mitochondrial genome has the following defects: First, there are mitochondrial pseudogenes in the nuclear genome. If the amplification region of the primers is short, the primers are very easy to combine with the nuclear genomic DNA, resulting in non-specific amplification. , the experimental results cannot rule out the interference of pseudogenes; secondly, some genetic diseases are caused by the duplication or deletion of large fragments of mtDNA. Auxiliary diagnosis of etiology

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  • Primer pair and method used for whole genome amplification of human mitochondria
  • Primer pair and method used for whole genome amplification of human mitochondria
  • Primer pair and method used for whole genome amplification of human mitochondria

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Embodiment Construction

[0032] Below in conjunction with accompanying drawing, the present invention is explained in detail.

[0033] The following steps are used to extract human whole gene DNA:

[0034] (1) Dispense 400 μL of erythrocyte lysate (component of Blood Genomic DNA Extraction Kit of Tiangen Biochemical Technology Co., Ltd.) into a 1.5 mL centrifuge tube, add 200 μL of whole blood, mix well; incubate at 56°C for 10 min;

[0035] (2) Centrifuge at 13000 g for 1 min, discard the supernatant, add 200 μL red blood cell lysate, mix well, add 10 μL Protease K (Tiangen Biochemical Technology Co., Ltd.), mix well;

[0036] (3) Centrifuge at 13,000 g for 1 min, transfer the supernatant to a clean 1.5 mL centrifuge tube, add 400 μL of isopropanol, mix well, and DNA precipitates can be seen after mixing;

[0037] (4) Centrifuge at 13,000 g for 1 min, discard the supernatant, and add 400 μL of 70% ethanol to rinse the precipitate; repeat the operation once;

[0038] (5)) Open the lid to completely ...

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Abstract

The invention provides a primer pair used fro whole genome amplification of human mitochondria. The primer pair is shown in the following:mtDNA:F:ACTTTTAACCAGTGAAATTGACCTGCC:mtDNA:R:AAGAGACAGCTGAACCCTCGTGG. Compared with segmented amplification, the adopted method for total-ring amplification through the primer pair has the advantage that whole a detecting point mutates, large-segment repetition and missing of mitochondrion DNA can be found.

Description

technical field [0001] The invention relates to gene amplification technology, in particular to a whole genome amplification method of human mitochondria. Background technique [0002] Mitochondrial DNA (mtDNA) is a cell's second set of genetic material. Human mtDNA has a total of 16569 nucleotides and is double-stranded circular, including 37 genes encoding 13 proteins. [0003] mtDNA has a higher mutation rate than nuclear DNA and lacks repair capacity. Mutations in mtDNA can cause a series of genetic diseases, such as Leber hereditary optic neuropathy (LHON), myoclonic epilepsy with ragged red muscle fibers (MERRF), and so on. By selecting appropriate primers, the entire circle of the mitochondrial genome was amplified and sequenced to search for possible pathogenic mtDNA mutations. [0004] The technology of using multiple pairs of primers to amplify the whole circle of the mitochondrial genome has the following defects: First, there are mitochondrial pseudogenes in t...

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Application Information

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IPC IPC(8): C12N15/11C12N15/10
CPCC12N15/10C12N15/11C12Q2531/113
Inventor 刘立群赵苏州戴婵
Owner 北京信诺佰世医学检验所有限公司
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