Method for amplifying o complete sequence of mitochondria of golden coin turtle by using DNA extracted from nails

A technique of three-line box turtle and mitochondria

Inactive Publication Date: 2014-06-18
PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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  • Abstract
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  • Claims
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Problems solved by technology

However, since the problem of extracting DNA by non-destructive sampling has not been resolved, the relevant research on it has been in its infancy, and even the complete mitochondrial sequence of the three-lined box turtle has not been reported so far.

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  • Method for amplifying o complete sequence of mitochondria of golden coin turtle by using DNA extracted from nails
  • Method for amplifying o complete sequence of mitochondria of golden coin turtle by using DNA extracted from nails
  • Method for amplifying o complete sequence of mitochondria of golden coin turtle by using DNA extracted from nails

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Embodiment 1

[0029] The method provided in this example to amplify the full-length mitochondria of the box turtle using toenails to extract DNA comprises the following steps:

[0030] (1) Toenail sampling

[0031] Use surgical scissors to cut the toenails of the box turtle, soak them in absolute ethanol and store them at -20°C until use;

[0032] (2) Extraction of genomic DNA

[0033] Before DNA extraction, toenails were soaked in 1×PBS buffer at 4°C overnight, and washed 2-3 times during this period. Take about 10-20 mg of each toenail sample, and use the MicroElute Genomic DNA Kit (OMEGA, USA) kit to extract DNA. ① Add the toenail sample to a 1.5mL centrifuge tube, and add 210-220 μL of TLBuffer; ② Cut the toenail sample as much as possible , add 20 μL of OB solution; ③ place in a shaker air bath with a temperature of 60-70 rpm / min, digest at 56-60 ° C for 3-5 hours; ④ centrifuge at 20,000xg for 2 min, and take the supernatant; Buffer, mix in 70°C water bath for 10 minutes; ⑥Add 220 μ...

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Abstract

The invention discloses a method for amplifying complete sequence of mitochondria of golden coin turtle by using a DNA (Deoxyribo-Nucleic Acid) extracted from nails. The method comprises the following steps of (1) selecting the nails of the golden coin turtle, pretreating and then extracting the DNA of the golden coin turtle by using a MicroElute Genomic DNA Kit, (2) designing 16 pairs of primers capable of covering the whole genome of the mitochondria of golden coin turtle; by taking the golden coin turtle DNA extracted and obtained in the step (1) as a template, carrying out PCR (Polymerase Chain Reaction) amplification by using the 16 pairs of primers, sequencing and splicing the PCR amplification products, thus obtaining the complete sequence of the mitochondria of golden coin turtle by amplification for the first time. The method is simple in steps and low in cost, is of non-invasive sampling and is capable of obtaining the complete sequences of the golden coin turtle mitochondria.

Description

technical field [0001] The invention belongs to the technical field of the three-lined box turtle, and in particular relates to a method for amplifying the full-length mitochondria of the three-lined box turtle by extracting DNA from toenails. Background technique [0002] Turtles are the oldest and most specialized reptiles. The earliest record of appearance on the earth is the Late Triassic, which has a history of about 220 million years. They are called "living fossils". In recent decades, due to illegal hunting and habitat loss, more and more turtle species are at risk around the world, and most of them have been included in the appendix of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). In Asia in particular, victims of the trade in food, traditional medicines and pets, most turtle members have all but disappeared in the wild, sparking the phenomenon of international concern known as the Asian Turtle Crisis. In 2011, the Int...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 朱新平李伟赵建洪孝友
Owner PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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