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A method for amplifying the full-length mitochondria of the three-lined box turtle by extracting DNA from toenails

A technology for three-line turtle and mitochondria, which is applied in the field of amplifying the full-length mitochondria of three-line turtle, can solve the problem that the complete mitochondrial sequence of three-line turtle has not been reported, and achieves strong repairability, long transportation time and cost saving. Effect

Inactive Publication Date: 2016-01-20
PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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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.

Method used

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  • A method for amplifying the full-length mitochondria of the three-lined box turtle by extracting DNA from toenails
  • A method for amplifying the full-length mitochondria of the three-lined box turtle by extracting DNA from toenails
  • A method for amplifying the full-length mitochondria of the three-lined box turtle by extracting DNA from toenails

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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 extract DNA with the MicroEluteGenomicDNAKit (OMEGA, USA) kit. ① Add the toenail sample to a 1.5mL centrifuge tube, add 210-220 μL of TLBuffer; ② Cut the toenail sample as much as possible, and add 20 μL OB solution; ③ placed in a shaker air bath with a temperature of 60-70 rpm / min, digested at 56-60°C for 3-5 hours; ④ centrifuged at 20,000xg for 2 min, and took the supernatant; ⑤ added 220 μL of BLBuffer, mixed Water bath at 70°C for 10 minu...

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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). Particularly in Asia, victims of the trade in food, traditional medicines and pets, most members of the turtle have all but disappeared in the wild, sparking the phenomenon of international concern known as the Asian Turtle Crisis. In 2011, t...

Claims

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Application Information

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Patent Type & Authority Patents(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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