CN1718728A

CN1718728A - Extraction method of Pu goa feaces DNA

Info

Publication number: CN1718728A
Application number: CN 200410062721
Authority: CN
Inventors: 李迪强; 洪艳芸
Original assignee: Individual
Current assignee: Individual
Priority date: 2004-07-07
Filing date: 2004-07-07
Publication date: 2006-01-11

Abstract

A process for extracting the DNA of Przhevalskis protoantelope from its dung includes such steps as taking less dung, suspending it in physiological saline, centrifugal separation of supernatant, adding lysozyme, centrifugal separation of deposit, adding the mixed solution of Tris-HCl, EDTA and NaCl containing SDS and proteinase, water bath, extracting twice to obtain DNA specimen, washing with the solution of glacial acetic acid twice, vacuum drying dissolving in buffer liquid and storing at -20 deg.C.

Description

The extracting method of Pu goa feaces DNA

Affiliated technical field

The present invention relates to the extracting method of Pu goa feaces DNA, be specially adapted to the DNA analysis of Procapra przewalskii, also can be used for fields such as Procapra przewalskii population analysis, individual recognition and quantity surveying.

Background technology

Procapra przewalskii is classified as in imminent danger grade by world nature and natural conservation alliance (IUCN), belongs to country-level focused protection wildlife, domestic wild Procapra przewalskii quantity seldom, population quantity is about 300.Though the population quantity of Procapra przewalskii has increase trend, because fenced grassland makes the Procapra przewalskii habitat become incoherent, living environment constantly worsens.The Procapra przewalskii population has been separated into several little isolated populations now, and there is not the corridor, habitat between them, this makes that their may inbreeding, thereby cause the inbreeding depression in the heredity, add genetic drift, make Procapra przewalskii part individual adaptability seriously descend, thereby make the population viability of whole Procapra przewalskii species seriously descend.

For the ease of Procapra przewalskii is carried out genetics research; particularly carry out the research of genetic diversity, individual recognition and paternity test and the quantity surveying etc. of Procapra przewalskii; for the protection of Procapra przewalskii population provides the genetics foundation, need to extract Procapra przewalskii and get the DNA sample.

For non-damage on one's body animal extracts DNA, " Northeast Forestry University's journal " the 28th volume the 7th phase 72-77 page or leaf, " heredity " the 23rd volume the 3rd phase 217-219 page or leaf, " Shandong animal and veterinary " the 2nd phase 1-2 page or leaf in 1999 and Chinese patent 97107478 etc. had once proposed to extract from animal hairs such as Zhu, giant pandas the method for DNA respectively, carried out the population analysis, sex identification of animal etc.Animal body need be caught or directly be contacted to these methods, and there is certain degree of difficulty naturally in the acquisition of material.So these methods have some limitations in some particular animal species or object research field." animal journal " 2003 the 49th volumes the 5th phase 670-674 page or leaf, " economic animal journal " 2003 the 7th volumes the 1st phase 32-34 page or leaf have then proposed from extract the method for DNA from giant panda ight soil.More external academic journals have also been delivered some methods from animal excrement extraction DNA, roll up 225-234 pages or leaves 23 as " Molecular Ecology " 1997 6 volumes 225-234 page or leaf, 483-486 page or leaf, 1091-1097 page or leaf and " Nucleic Acids Research " nineteen ninety-fives 6 and roll up 18 phase 3800-3801 pages or leaves.

The Procapra przewalskii running speed is fast, is difficult for catching capturing, and general DNA extraction material is difficult to obtain, and the acquisition of its ight soil is then much easier.Yet because the biochemical substances that contains such as inhibitory substance that some is specific are different with other animals in the Pu goa feaces, so the Pu goa feaces DNA extracting method also is different from other animals.The present invention is a material with the ight soil of Procapra przewalskii, utilizes conventional biochemical reagents, therefrom extracts DNA, is used for researchs such as the population analysis of Procapra przewalskii and individual recognition.

Summary of the invention

For Procapra przewalskii is carried out DNA analysis, the invention provides a kind of extracting method of Pu goa feaces DNA.

This method is to utilize conventional biochemical reagents, after dissolving, centrifugal, precipitation and drying, obtains high-quality DNA at low cost from ight soil.Concrete technical scheme is: the ight soil of getting 0.2 gram cryopreservation is suspended in the physiological saline of 2ml 0.8%, and 4 ℃ of low-speed centrifugals 5 minutes are got supernatant and obtained solution A; Add N,O-Diacetylmuramidase in A, room temperature was placed 5-7 minute, and centrifugal 5 minutes of middling speed is got precipitation and obtained B; In B, add the mixed solution that 0.5ml contains Tutofusin tris hydrochloric acid (Tris-HCl), ethylenediamine tetraacetic acid (EDTA) (EDTA), sodium-chlor (NaCl), wherein also contain Sodium dodecylbenzene sulfonate (SDS) and Proteinase K, obtain solution C; 55 ℃ water-bath 3.5-4 hour; Then solution C is carried out 2 extractings: in C, add the saturated phenol of equal-volume for the first time, middling speed after centrifugal 10 minutes the extracting supernatant liquor obtain solution D, in D, add the equal-volume chloroform for the second time, middling speed after centrifugal 10 minutes the extracting supernatant liquor obtain solution E, add the sodium acetate soln of 1/10 volume and the ice dehydrated alcohol of 2.5 times of volumes in E ,-20 ℃ are spent the night; High speed centrifugation obtains the DNA sample F after 15 minutes; Use 70% ice washing with alcohol F twice at last, be dissolved in behind the vacuum-drying F in an amount of damping fluid in-20 ℃ of preservations.

Embodiment

Present method embodiment is: the ight soil of getting 0.2 gram cryopreservation is suspended in the physiological saline of 2ml 0.8%, and 4 ℃ of low-speed centrifugals 5 minutes are got supernatant and obtained solution A; Add N,O-Diacetylmuramidase in A, room temperature was placed 5-7 minute, and centrifugal 5 minutes of middling speed is got precipitation and obtained B; In B, add the mixed solution that 0.5ml contains Tutofusin tris hydrochloric acid (Tris-HCl), ethylenediamine tetraacetic acid (EDTA) (EDTA), sodium-chlor (NaCl), wherein also contain Sodium dodecylbenzene sulfonate (SDS) and Proteinase K, obtain solution C; 55 ℃ water-bath 3.5-4 hour; Then solution C is carried out 2 extractings: in C, add the saturated phenol of equal-volume for the first time, middling speed after centrifugal 10 minutes the extracting supernatant liquor obtain solution D, in D, add the equal-volume chloroform for the second time, middling speed after centrifugal 10 minutes the extracting supernatant liquor obtain solution E, add the sodium acetate soln of 1/10 volume and the ice dehydrated alcohol of 2.5 times of volumes in E ,-20 ℃ are spent the night; High speed centrifugation obtains the DNA sample F after 15 minutes; Use 70% ice washing with alcohol F twice at last, be dissolved in behind the vacuum-drying F in an amount of damping fluid in-20 ℃ of preservations.

Claims

1. the extracting method of Pu goa feaces DNA is characterized in that: the ight soil of getting 0.2 gram cryopreservation is suspended in the physiological saline of 2ml 0.8%, and 4 ℃ of low-speed centrifugals 5 minutes are got supernatant and obtained solution A; Add N,O-Diacetylmuramidase in A, room temperature was placed 5-7 minute, and centrifugal 5 minutes of middling speed is got precipitation and obtained B; In B, add the mixed solution that 0.5ml contains Tutofusin tris hydrochloric acid (Tris-HCl), ethylenediamine tetraacetic acid (EDTA) (EDTA), sodium-chlor (NaCl), wherein also contain Sodium dodecylbenzene sulfonate (SDS) and Proteinase K, obtain solution C; 55 ℃ water-bath 3.5-4 hour; Then solution C is carried out 2 extractings: in C, add the saturated phenol of equal-volume for the first time, middling speed after centrifugal 10 minutes the extracting supernatant liquor obtain solution D, in D, add the equal-volume chloroform for the second time, middling speed after centrifugal 10 minutes the extracting supernatant liquor obtain solution E, add the sodium acetate soln of 1/10 volume and the ice dehydrated alcohol of 2.5 times of volumes in E ,-20 ℃ are spent the night; High speed centrifugation obtains the DNA sample F after 15 minutes; Use 70% ice washing with alcohol F twice at last, be dissolved in behind the vacuum-drying F in an amount of damping fluid in-20 ℃ of preservations.