CN1052728C

CN1052728C - Method for extracting hologenome of giant panda

Info

Publication number: CN1052728C
Application number: CN97107478A
Authority: CN
Inventors: 方盛国; 冯文和; 张安居
Original assignee: Sichuan Normal University
Current assignee: Sichuan Normal University
Priority date: 1997-05-05
Filing date: 1997-05-05
Publication date: 2000-05-24
Anticipated expiration: 2017-05-05
Also published as: CN1198439A

Abstract

The present invention provides a method for extracting the complete genome DNA of a giant panda. A DNA extraction material is dewatered by using ethanol with different concentrations step by step, and processed through precipitate separation, critical drying, ethanol or formalin removal, pulverization., digestion by adding protease and SDS, extraction by using phenol and chloroform, precipitation by using cold ethyl alcohol and vacuum drying. The method achieves the sources of a DNA material form a giant panda and other wild and stable breeding animals, can be used for the research on the population inheritance of animal populations, and can be used for the gene analysis of a fixed test material of medically difficult and complicated diseases.

Description

Method for extracting hologenome of giant panda

The present invention relates to DNA gene extracting method, particularly relate to a kind of method for extracting hologenome of giant panda.

Domestic and international at present formalin and paraffin embedding organ tissue to animal, the DNA extraction of materials such as outmoded sample skin, only can obtain the DNA fragment about 1.0kb, be used for pcr analysis, can not obtain the complete genome DNA of animal, carry out the research of aspects such as Soutliern trace, genome analysis and clone.Meanwhile, utilize ethanol fixedly to appear in the newspapers at the research end of material extraction animal gastrointestinal tract exuviation histocyte complete genome DNAs such as organ-tissue, fresh excreta and urine, ethanol or formalin fixed ight soil.In recent years, progressively carry out for the analysis and research of giant panda DNA both at home and abroad, but research material all is taken from fresh blood, seminal fluid, skin and the hair of giant panda.Based on Daxiang Ling; the giant panda of two mountain systems in little phase mountain range; only there is the part individuality to live in the field at present; and Mount Min; the Liangshan Mountain; the giant panda of four big mountain systems such as the Qionglai and the Qinling Mountains; though being arranged, the part individuality lives in the wilderness area feedlot; zoological park and giant panda breed the study base; but because of individual amount few; and for many reasons such as protection rare species; if with fresh or refrigerated blood; seminal fluid; skin etc. are organized as the DNA material source; then extremely difficult; therefore; with the fixing organ-tissue of the formalin of giant panda in sample shop and the laboratory or ethanol; paraffin-embedded tissue; outmoded sample skin; even the fresh excreta of field or Captive Giant Panda; urine; formalin or ethanol fixedly ight soil etc. are the DNA material source, will be to solve the new way that experiment material is difficult to obtain in the research of giant panda DNA analysis.

The object of the present invention is to provide a kind of fresh excreta or urine that can be giant panda, formalin fixed organ-tissue or fixing ight soil, ethanol is organ-tissue or ight soil fixedly, and samples such as paraffin embedding organ tissue and outmoded sample skin are the method for extracting hologenome of giant panda of analysis of material.

Method for extracting hologenome of giant panda of the present invention is:

1) extracts at hologenome of giant panda that the ratio in 1: 1 adds 80% ethanol in the material (fresh excreta, fire pot baking ight soil, formalin fixed ight soil, ethanol fixedly ight soil, urine, formalin fixed organ-tissue, ethanol fixedly organ-tissue, paraffin embedding organ tissue, outmoded sample skin), room temperature left standstill 0.5 hour, 3000 rev/mins centrifugal 5 minutes, separate, get precipitation.But when the DNA extraction material is ight soil and urine, should carries out two times centrifugal and separate, promptly add 80% ethanol, room temperature left standstill 0.5 hour, 3000 rev/mins centrifugal 5 minutes, after the separation, get supernatant liquid earlier, purpose is to remove the excrement slag, again with supernatant liquid once more 4000 rev/mins centrifugal 5 minutes, separate, virus and bacterium are fixed in the supernatant liquid, no longer produce enzyme and decompose giant panda DNA, abandon supernatant liquid, get precipitation at last.

2) 1) add 90% ethanol in the gained precipitation, room temperature left standstill 0.5 hour, 3000 rev/mins centrifugal 10 minutes, separate, get precipitation.

3) 2) add 100% ethanol in the gained precipitation, room temperature left standstill 0.5 hour, 3000 rev/mins centrifugal 10 minutes, separate, get precipitation.

4) with 3) gained is precipitated in the filter paper bag, seals, and puts into the critical evaporator sample chamber, adds liquid carbon dioxide by steel cylinder, surpass the paper bag vertex to the liquid carbon dioxide liquid level till.

5) heat after 31 ℃ (normal atmosphere 72.8) in the sample chamber, kept two hours, opens the venting valve, discharges gas, thoroughly removes formalin and ethanol simultaneously, takes out paper bag, gets pure dry sample.

6) sample in the paper bag is put into mortar, add liquid nitrogen, sample is freezing after 0.2 minute becomes fragile, grind and be powder, add the TNE damping fluid, move in the centrifuge tube in 1: 1 ratio, add Proteinase K and SDS, make final concentration be respectively 50mg/l and 0.5% (weight), in 55 ℃ of insulation digestion 3 hours.

7) carry and take out 2 times with heavily steaming phenol, remove deproteinize and bacterium, carry with chloroform and taking out once, remove phenol, add 95% cold ethanol (10 ℃) precipitation, with big mouthful of suction pipe sucking-off hologenome of giant panda, vacuum-drying 1 hour is preserved standby down in 4 ℃.

The advantage of method for extracting hologenome of giant panda of the present invention is that complete genome DNA extracts material and can be fresh excreta, fire pot baking ight soil, formalin fixed ight soil, ethanol is ight soil fixedly, urine, the formalin fixed organ-tissue, ethanol is organ-tissue fixedly, paraffin embedding organ tissue, outmoded sample skin etc., this DNA can be used for the giant panda gene fingerprint analysis, also be used for simultaneously vertebrates (fish, batrachians, birds, mammals) and in human molecular biological applications and the fundamental research, as assert size of animal, family structure is formed, sex structure, mating behavior etc., obtain the population genetics quantitative data of population thus, be the dynamic management of wildlife, set up unique and be quantitative molecular genetics archives; Research has been died out or evolutionary biology, the molecular genetics of the animal that is on the brink of to die out; Medically, for the ethanol or the formalin fixed sample of some difficult and complicated illness that can not make a definite diagnosis in the past, available this method is extracted complete genome DNA, carries out genetic analysis and diagnosis; In addition, in forensic science is used, utilize this technological method, from the on-the-spot micro-sample of suspect, extract complete genome DNA, make fingerprint detection, assert criminal by the genetic fingerprint probe.

Describe the embodiment of the invention below in detail.

Embodiment 1: get outmoded sample skin 0.5 gram, adds 0.5 milliliter of 80% ethanol, room temperature left standstill 0.5 hour, 3000 rev/mins centrifugal 5 minutes, precipitation is got in separation.Add 0.5 milliliter of 90% ethanol in gained precipitation, room temperature left standstill 0.5 hour, 3000 rev/mins centrifugal 10 minutes, separate, get precipitation.Add 0.5 milliliter of 100% ethanol in gained precipitation, room temperature left standstill 0.5 hour, 3000 rev/mins centrifugal 10 minutes, separate, get precipitation.Gained is precipitated in the filter paper bag, seals, put into the critical evaporator sample chamber, add liquid carbon dioxide by steel cylinder, surpass the paper bag vertex to the liquid carbon dioxide liquid level till.Heating after 31 ℃ (normal atmosphere 72.8) in the sample chamber, kept two hours, opens the venting valve, drains liquid carbon dioxide, takes out paper bag.Sample in the paper bag is put into mortar, add about 10 milliliters of liquid nitrogen, 0.2 grind sample after minute to Powdered, add 0.5 milliliter of TNE damping fluid (10mmol Tris-10mmol EDTA-100mmol NaCL, PH7.8) move in the centrifuge tube, add Proteinase K and SDS, make final concentration be respectively 50mg/l and 0.5% (weight), in 55 ℃ of insulation digestion 3 hours.Add 2 milliliters and heavily steam phenol, swayed 5 minutes, 3000 rev/mins centrifugal 5 minutes, get upper strata liquid, add 2 milliliters of phenol, repeat above-mentioned swaying and centrifugal process, get upper strata liquid, add 2 milliliters of chloroforms, repeat above-mentioned swaying and centrifugal process, get upper strata liquid, add 10 milliliter of 95% cold ethanol (10 ℃) precipitation, shook about 1 minute, with big mouthful of suction pipe sucking-off complete genome DNA, vacuum-drying 1 hour is preserved standby in 4 ℃ of refrigerators.

Embodiment 2: get paraffin embedding testis organ-tissue 0.5 gram, adds 5 milliliters of dimethylbenzene, vortex vibrated 0.5 minute, 4000 rev/mins centrifugal 5 minutes, abandon supernatant liquor, get precipitation, following steps are with embodiment 1.

Embodiment 3: the basin of getting fire is dried ight soil 0.5 gram, add 0.5 milliliter and add 80% ethanol, room temperature left standstill 0.5 hour, 3000 rev/mins centrifugal 5 minutes, after the separation, get supernatant liquid earlier, once more 4000 rev/mins centrifugal 5 minutes, separate, virus and bacterium are fixed in the supernatant liquid, abandon supernatant liquid, get precipitation at last.Following steps are with embodiment 1.

Above-mentioned three embodiment gained DNA samples carry out fingerprinting with probe, and gene fingerprint is Fig. 1 and Fig. 2, shows that the hologenome of giant panda that is extracted is a pure dna.

Claims

1, a kind of method for extracting hologenome of giant panda is characterized in that may further comprise the steps:

1) ratio in 1: 1 adds 80% ethanol in hologenome of giant panda extraction material,

Room temperature leaves standstill, and precipitation is got in centrifugation;

2) 1) add 90% ethanol in the gained precipitation, room temperature leaves standstill, and precipitation is got in centrifugation;

3) 2) add 100% ethanol in the gained precipitation, room temperature leaves standstill, and precipitation is got in centrifugation;

4) with 3) gained is precipitated in the filter paper bag, seals, and puts into the critical evaporator sample chamber, by steel cylinder

Add liquid carbon dioxide, surpass the paper bag vertex to the liquid carbon dioxide liquid level till;

5) heat to 31 ℃ in the sample chamber, after normal atmosphere is 72.8 mmhg, kept two hours,

Open the venting valve, discharge gas, take out paper bag;

6) sample in the paper bag is put into mortar, add liquid nitrogen, grind and be powder, by 1: 1 ratio adding T

The NE damping fluid moves in the centrifuge tube, adds Proteinase K and SDS, in 55 ℃ of insulations

Digestion;

7) carry with phenol respectively and taking out, chloroform is carried and being taken out, 95% cold ethanol precipitation, the full genome of sucking-off giant panda

DNA, vacuum-drying is preserved standby down in 4 ℃;

Wherein, to extract material be fixedly fixedly organ-tissue, paraffin embedding organ tissue, outmoded sample skin of ight soil, urine, formalin fixed organ-tissue, ethanol of fresh excreta, fire pot baking ight soil, formalin fixed ight soil, ethanol to hologenome of giant panda.

2, method for extracting hologenome of giant panda as claimed in claim 1, when it is characterized in that the DNA extraction material is ight soil and urine, should carry out two times centrifugal separates, promptly add 80% ethanol, room temperature leaves standstill, and after the centrifugation, gets supernatant liquid earlier, again the supernatant liquid recentrifuge is separated, get precipitation at last.

3, method for extracting hologenome of giant panda as claimed in claim 1 is characterized in that the DNA extraction material is a paraffin embedding organ when organizing, and needs dewax with dimethylbenzene earlier.

4, method for extracting hologenome of giant panda as claimed in claim 1 is characterized in that centrifuge speeds is 3000～4000 rev/mins.

5, method for extracting hologenome of giant panda as claimed in claim 1, it is characterized in that adding Proteinase K and SDS after, make final concentration be respectively 50mg/l and 0.5% (weight).