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Method for amplifying complete sequence of mitochondrial genome of Macrobrachium nipponense

A technology of mitochondrial genome and mitochondrial gene, which is applied in the field of full-sequence amplification of the mitochondrial genome of Macrobrachium japonicus, and the field of full-sequence amplification of the mitochondrial genome, which can solve the problem of low yield of extracted mitochondrial genomic DNA, difficulty in repeating mitochondrial DNA in experiments, long operation time, etc. problems, to achieve the effect of not easy to degrade, the requirements of experimental conditions are not strict, and the operation time is short

Inactive Publication Date: 2011-04-13
SHANGHAI OCEAN UNIV
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Problems solved by technology

[0006] The present invention is in order to solve the above-mentioned problem, overcomes such as Triton method, alkali denaturation method, proteinase K method, but all has shortcoming: Triton method extracts the output of mitochondrial genomic DNA low, and easily degrades; The experimental condition that alkali denaturation method requires is stricter, The experiment is not easy to repeat and the extracted mitochondrial DNA may have two structures: circular and open; the proteinase K method requires a long operation time, etc., providing a method for the full sequence amplification of the macrobrachium mitochondrial genome

Method used

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  • Method for amplifying complete sequence of mitochondrial genome of Macrobrachium nipponense
  • Method for amplifying complete sequence of mitochondrial genome of Macrobrachium nipponense
  • Method for amplifying complete sequence of mitochondrial genome of Macrobrachium nipponense

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Embodiment

[0046] Experiment 1. Extraction of the mitochondrial genome of Macrobrachium japonicus by high-salt precipitation method

[0047] Use liquid nitrogen to grind fresh giant river prawn (Macrobrachium nipponense) muscles collected in Shanghai, China to powder, transfer 50 mg to a 1.5 mL centrifuge tube and add solution I (Tris-HCl 10 mmol / L, NaCl 10 mmol / L, MgCl 2 5mmol / L pH 7.5) 500μl, mix well, 2000r / min, 10min, discard the supernatant. Add solution II (Tris-HCl 10mmol / L, NaCl 400mmol / L, EDTA 2mmol / L pH 8.0) 500μl to the precipitation, mix well, 3500r / min, 10min, discard the supernatant. Precipitation plus solution II 100 μl, after mixing, add 5 μl of 20 mg / mL proteinase K and 20 μl of 10% SDS, and mix quickly. 55°C water bath for 1.5h. Add 50 μl saturated sodium acetate, mix well, and shake gently for 15S. Centrifuge at 15000r / min, 4°C for 15min, and take the supernatant into an Eppendrof tube. Add 50 μl saturated sodium acetate again, mix well, and shake gently for 15S. ...

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Abstract

The invention discloses a method for amplifying a complete sequence of a mitochondrial genome of a Macrobrachium nipponense. The method comprises the following steps of: extracting the mitochondrial genome of the Macrobrachium nipponense by a high-salinity precipitation method; amplifying two gene segments, namely cytochrome oxidase I (CO I) and 16S rRNA in the mitochondrial genome of the Macrobrachium nipponense; and performing long polymerase chain reaction (PCR) amplification on the mitochondrial genome deoxyribose nucleic acid (DNA) of the Macrobrachium nipponense. Mitochondrial DNA (mtDNA) is a very useful material in the research of molecular genetic evolution. As a mitochondrial gene is not rearranged during cell reduction division and the point mutation rate is high, the method contributes to examining the changes of the gene in a shorter period and comparing the difference among the same genes of different species so as to determine the evolutionarily genetic relationship of the species.

Description

technical field [0001] The invention relates to a method for amplifying the full sequence of the mitochondrial genome, in particular to a method for amplifying the full sequence of the mitochondrial genome of Macrobrachium japonicus, and belongs to the field of molecular biology technology. Background technique [0002] Macrobrachium nipponense (Macrobrachium nipponense), commonly known as freshwater shrimp, belongs to the class Crustacea, Decapod order, Longarm shrimp family, Macrobrachium genus. Macrobrachium japonicus has the characteristics of strong adaptability, wide distribution, miscellaneous food habits, fast growth, and high economic benefits of breeding. It is the most important freshwater economic shrimp in my country. At present, most of the research work on Macrobrachium japonicus focuses on growth characteristics, karyotype analysis, and seedling breeding, while there are relatively few studies on Macrobrachium japonicus using molecular biology methods, mainly...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 李家乐马克异冯建彬姜虎成
Owner SHANGHAI OCEAN UNIV
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