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Fish mitochondria genome-wide DNA amplification

A whole genome, mitochondrial technology, applied in the fields of genetics and molecular biology, can solve the problems of high requirements and high cost of whole genome assembly technology, and achieve the effect of simplifying PCR and purification steps

Inactive Publication Date: 2015-09-16
CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this method requires high-depth genome sequencing and high cost; at the same time, it requires high technology for whole genome assembly; the results of sequencing and assembly are mainly nuclear genomic DNA

Method used

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  • Fish mitochondria genome-wide DNA amplification
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  • Fish mitochondria genome-wide DNA amplification

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Embodiment 1

[0048] See Table 1 for the PCR primer pairs used for mitochondrial whole genome sequencing of the present invention.

[0049] The PCR amplification process is:

[0050] For mitochondrial genome sequencing, the above four pairs of PCR primers including all combinations of A, T, C, and G were selected, and the upstream and downstream primers were diluted to 10 μmol / L as the working solution. PCR reaction system: The total volume of the system is 20 μL, which contains 1 μL of DNA template (including at least 50 ng of whole genome DNA), 0.5 μL of the corresponding upstream and downstream primers (10 μmol / L) of the target fragment, 10 μL of 2×Taq enzyme reaction mixture (reaction The mixture includes 0.1U / μL LA-Taq enzyme, 2×PCR reaction buffer, 3mmol / L MgCl2, and 0.4mmol / L dNTP), ddH2O 8μl.

[0051] Wherein, the aforementioned DNA templates are respectively the genomic DNAs of penguin lamp, steelhead trout, rainbow trout, crucian carp, golden trout and carp, and the method for ex...

Embodiment 2

[0057] In order to verify whether the amplification results are correct, we selected the amplification products of penguin lamp and steelhead trout to be sequenced respectively. We purified the PCR amplified products and performed Sanger sequencing. The 4-segment sequencing results of Penguin Lamp were spliced ​​and assembled to obtain a complete mitochondrial genome sequence, the sequence of which is shown in sequence 9 in the sequence listing. The assembly of steelhead trout mitochondrial genome sequence was also performed as above, and its sequence is shown in sequence 10 in the sequence listing.

[0058] The length of the whole mitochondrial genome sequence of Penguin lamp is 16524bp, which is consistent with the length interval of most fish mitochondrial genomes. This sequence is the first published complete mitochondrial genome sequence of Penguin lamp. Homologous sequence comparison found that the most similar sequence to this sequence was the mitochondria of the blin...

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Abstract

The invention provides fish mitochondria genome-wide DNA amplification. The invention firstly provides a primer pair for fish mitochondria genome-wide DNA amplification. The invention further provides a method for fish mitochondria genome-wide DNA amplification based on the primer combination. By the adoption of the amplification method, clone products of various fish mitochondria genomes can be obtained successfully. In addition, a primer pair obtained through designing and screening has the excellent characteristics that the amplification performance is high and the number of by-products generated is small.

Description

technical field [0001] The invention relates to the fields of genetics and molecular biology, in particular to a pair of primers for amplifying fish mitochondrial genomic DNA and an amplifying method. Background technique [0002] Fish mitochondrial genome (mtDNA) is a closed circular double-stranded DNA with autonomous replication, transcription and translation capabilities in the cytoplasm, including a heavy chain and a light chain. The fish mitochondrial genome mainly includes 37 coding genes (13 hydrophobin genes, 2 rRNA genes, 22 tRNA genes), a control region (D-loop) responsible for replication and transcription initiation, and a light chain replication starting area. The polypeptide encoded by the hydrophobin gene includes the subunits of the enzyme complex combined with the inner mitochondrial membrane: cytochrome b (Cyt b), two subunits of ATP synthase (ATPase6 and ATPase8), three cytochrome c ( Cyt c) subunits of oxidases (COX1, COX2 and COX3) and seven subunits ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/10
Inventor 李炯棠薛尉肖贵宝孙效文
Owner CHINESE ACAD OF FISHERY SCI
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