Reagent kit for extracting DNA in Chinese alligator chorion film and use method thereof

An eggshell membrane and kit technology, applied in the field of DNA extraction, can solve problems such as unfavorable research, unsuitable DNA extraction from non-damaging materials, etc., and achieve the effects of good purity, high concentration and strong specificity

Inactive Publication Date: 2009-02-18
ANHUI NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, most DNA extraction methods have strict requirements on sample materials and require sufficient sample size, which is not conducive to research in some special fields, such as DNA analysis of rare and endangered wild animals, species identificat

Method used

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  • Reagent kit for extracting DNA in Chinese alligator chorion film and use method thereof
  • Reagent kit for extracting DNA in Chinese alligator chorion film and use method thereof
  • Reagent kit for extracting DNA in Chinese alligator chorion film and use method thereof

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Experimental program
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Effect test

Embodiment 1

[0042] The reagents of the kit used in this embodiment are prepared according to the following formula:

[0043](a) Reagent A: a preservation solution, containing physiological saline, 100mmoL / L glucose, 100U / ml penicillin, and 100U / ml streptomycin;

[0044] (b) Reagent B: for pretreatment liquid, comprise phosphate buffer (B1) and STE buffer (B2),

[0045] Described phosphate buffer (B1): containing 137mM NaCl; 2.7mM KCl; 10mM NaCl 2 HPO 4 ; 2mM KH 2 PO 4 ;

[0046] The STE buffer (B2): contains 1M Tris-HCl pH8.0, 0.5M EDTA, 5M NaCl;

[0047] (c) Reagent C: lysate 1, containing 50mM Tris-HCl pH8.0, 1mM EDTA pH8.0, 150mM NaCl, 1mol / L dithiothreitol (DTT), mass concentration of 20% dodecylbenzene Sodium sulfonate (SDS), 20mg / ml proteinase K;

[0048] (d) Reagent D: lysate 2, containing 20 mg / ml proteinase K and a mass concentration of 0.2% β-mercaptoethanol;

[0049] (e) Reagent E: extract 1, containing saturated balanced phenol, chloroform, isoamyl alcohol, and its vol...

Embodiment 2

[0061] The reagents of the kit used in this embodiment are prepared according to the following formula:

[0062] (a) Reagent A: a preservation solution, containing physiological saline, 100mmoL / L glucose, 100U / ml penicillin, and 100U / ml streptomycin;

[0063] (b) Reagent B: pretreatment solution, including phosphate buffer (B1) and STE buffer (B2),

[0064] Described phosphate buffer (B1): containing 135mM NaCl; 3.0mM KCl; 10mM NaCl 2 HPO 4 ; 1.5mM KH 2 PO 4 ;

[0065] The STE buffer (B2): contains 1.5M Tris-HCl pH8.0, 0.5M EDTA, 6M NaCl;

[0066] (c) Reagent C: lysate 1, containing 50mM Tris-HCl pH8.0, 0.5mM EDTA pH8.0, 100mM NaCl, 1mol / L mM dithiothreitol (DTT), the concentration (w / v) is 10% Sodium dodecylbenzenesulfonate (SDS), 20mg / ml proteinase K;

[0067] (d) Reagent D: lysate 2, containing 20 mg / ml proteinase K and a mass concentration of 1% β-mercaptoethanol;

[0068] (e) Reagent E: extract 1, containing saturated balanced phenol, chloroform, isoamyl alcohol, ...

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Abstract

The invention discloses a kit for extracting DNA in the egg shell membrane of Yangtze alligator and a use method thereof, and the kit comprises the following reagents: a reagent A, a reagent B, a reagent C, a reagent D, a reagent E and a reagent F. The template DNA which is extracted by the kit has good purity, and the kit can be used for gene amplification sequencing and typing. In the amplification of the 12S rRNA gene, the mitochondrial D-loop region and the Cyt b gene, all the extracted shell membrane DNA templates can be successfully amplified to obtain the fragment products with expected identical sizes, high concentration and high specificity. Microsatellite DNA marker specificity primers are utilized to obtain typing data through the PCR amplification to be used for the identification in disputed paternity of Yangtze alligator. In addition, the method also can be applied to the extraction of other ovipara such as the turtles, the birds and the like. The obtained template DNA can be widely applied to the studies of the ovipara on the fields such as population genetic analysis, molecular evolution, evolution of relationship among individuals and the like.

Description

technical field [0001] The invention relates to a kit for extracting DNA from alligator eggshell membranes and a use method thereof. Background technique [0002] The alligator (Alligator sinensis) belongs to the class Reptiles, Crocodiles, Scorpidae, and the genus Sinensis. It is called a "living fossil" because it originated from the Mesozoic Era that was at the same time as the dinosaurs more than 200 million years ago. It is one of the 23 species of crocodiles in the world. One of the most endangered species in China, it is listed as critically endangered by the International Union for the Conservation of Nature and Natural Resources (IUCN), a CITES Appendix I species, and a national first-class protected wild animal. At present, the wild population of Chinese alligator is about 120-140, and it is still declining at a rate of 4%-6% per year. It is on the verge of extinction, and it is urgent to restore its gene flow artificially. The breeding population of Chinese allig...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/68
Inventor 吴孝兵胡芸晏鹏
Owner ANHUI NORMAL UNIV
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