Method for amplifying mitochondrial gene total sequences of fish in Yangtze River based on degenerate primer combination and application thereof

A mitochondrial gene and degenerate primer technology, applied in the field of molecular biology, can solve the problem of high cost

Pending Publication Date: 2018-01-09
CHINESE STURGEON RES INST CHINA THREE GOR
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The first method relies too much on closely r

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  • Method for amplifying mitochondrial gene total sequences of fish in Yangtze River based on degenerate primer combination and application thereof
  • Method for amplifying mitochondrial gene total sequences of fish in Yangtze River based on degenerate primer combination and application thereof
  • Method for amplifying mitochondrial gene total sequences of fish in Yangtze River based on degenerate primer combination and application thereof

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Experimental program
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Embodiment 2

[0101] Example 2 Known fish species verify the correctness of degenerate primer amplification results and the classification and identification of unknown fish species

[0102] 1. Sample Collection

[0103] The samples come from the fin ray samples collected by the Chinese Sturgeon Research Institute and the fish catch survey in the upper reaches of the Yangtze River. The fin rays were preserved in ethanol and brought back. If the time is long, the ethanol should be replaced regularly.

[0104] 2. Sample DNA Extraction

[0105] Use the TIANGEN TIANamp Genomic DNA Kit (blood / cell / tissue genomic DNA extraction) kit to extract the DNA of the species to be tested. When extracting, first cut the sample tissue (fins, muscles, fish eggs, etc.), pick a small amount of samples into 200 μl buffer GA, add 20 μl proteinase K solution, mix well, and digest at 56°C for 30-60 minutes. Add 200 μl buffer GB, mix thoroughly by inversion, and place at 70°C for 10 minutes. Add 200 μl of absolu...

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Abstract

The invention discloses a method for amplifying mitochondrial gene total sequences of fishes in Yangtze River based on a degenerate primer combination and application thereof. The primer combination is obtained by designing in a conserved region through comparing the mitochondrial total sequences of fishes of acipenseridae, salangidae, cyprinidae, salmonidae, anguillidae, siluridae, bagridae, cyprinodontidae, hemiramphidae, synbranchidae, serranidae, gobiidae, channidae, bagridae and cobitidae, and the primer combination comprises Mt-1F, Mt-1R; Mt-2F, Mt-2R; Mt-3F, Mt-3R; Mt-4F, Mt-4R; Mt-5F,Mt-5R; Mt-6F, Mt-6R; and Mt-7F, Mt-7R. According to the method, the seven pairs of primers are used for carrying out PCR amplification on corresponding gene fragments, and the mitochondrial gene totalsequences are obtained through connection transformation, sequencing and splicing; the primer combination is less, the amplification effect is good, and the monitored fishes and roe can be effectively classified and identified. According to method provided by the invention, the degenerate primers are designed on the basis of the fish mitochondria conserved region, so that the mitochondrial gene total sequences of various fishes can be obtained; the universality is high, the obtained data can be applied to the research of fish mitochondrial genomes, thus perfecting a mitochondria database.

Description

technical field [0001] The present invention relates to the field of molecular biology, and specifically refers to a method for amplifying the complete mitochondrial gene sequence of Yangtze River fish based on a combination of degenerate primers and its application Background technique [0002] Traditional classification of fish species often relies on their external morphological characteristics, including body length, height, body color, number of fin rays, markings, or relative measurements of body parts. However, some fishes have a wide distribution range, many species, and morphological characteristics change greatly in different growth stages, which has caused certain difficulties for researchers to identify fish species. In addition, some fish are similar in appearance in the early development stage, and some deviations often occur in species identification, and even professional researchers will inevitably make mistakes. At present, there are many ecological monito...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888
Inventor 李莎刘雪清姜伟
Owner CHINESE STURGEON RES INST CHINA THREE GOR
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