CN1904066B - Extraction method of mammal faeces DNA - Google Patents

Abstract

The present invention relates to a method for extracting DNA from mammalian excrement. Said method includes collection of excrement, storage, sampling and DNA extraction. The invented method is characterized by that said method includes the following steps: firstly, using lysis liquor containing cetyl trimethyl ammonium bromide to make DNA be released out from cell, then making separation to obtain DNA precipitate, finally using guanidinium thiocyanate and silicone dioxide to make adsorption and purification so as to obtain high-purity DNA.

Description

Extraction method of mammal faeces DNA

One, technical field

The present invention relates to the extracting method of animal DNA, particularly in non-damage sampling material, extract the method for DNA, specifically in mammal faeces, extract the method for DNA.

Two, background technology

In recent years, the application of molecular genetics in conservation biology and behavioral ecology is subjected to generally paying attention to, and the primary work of carrying out molecular genetics analysis is the collection of analytic sample.The sampling method in past mainly contains two kinds: the one, and nocuity sampling is promptly killed animal and is obtained tissue samples such as fresh muscle, liver and blood.This method can cause very big destruction to Animal resources; The 2nd, non-nocuity sampling is promptly extracted blood by catching animal, is gathered analysis samples such as hair or feather ear, tail or toe.Though this method is unlikely to cause animal dead, particularly habit and the viability of rare species all can impact to wildlife.

The non-damage sampling is not touch or do not injuring under the situation of wildlife itself, and the hair or the multi-form analytic samples such as feather, ight soil, urine, swill (containing the oral cavity cast-off cells), deer horn, fish scale and chorion that come off by collection carry out the extraction and the analysis of genetic material.In non-damage sampling material, animal excrement are a kind of ideal analytic samples, and reason comprises: one, the animal defecation has rhythmicity, and with respect to other non-damage sampling materials, the sample abundance is easy to gather; Two, fecal sample can obtain not contacting under the situation of even not seeing animal, to the interference of animal with injure for a short time, has also protected the personnel safety of field worker simultaneously when the large-scale zoophagous animal of research; Three, when the big species of population are sampled on a large scale, catch animal and sample, gather the cost that ight soil can significantly reduce sampling.Therefore, this method has been got rid of all unfavorable factors of traditional sampling method, has solved a sampling difficult problem that is run in protection genetics and the molecular ecology research basically.Successfully having been applied to species in recent years differentiates the identification (little satellite is increased) of (by the amplification to Mitochondrial DNA), individuality, animal sex identification (sry gene on the Y chromosome is increased), plants the evaluation (district increases to mitochondria control) of edge relation, maternal identify (by the amplification to Mitochondrial DNA), paternity test (little satellite is increased), population quantity estimation (little satellite is increased) or the like (Kohnand Wayne, 1997).

In these researchs, the collection of faecal samples and the progress of the sampling method before store method and the extraction thereof have been promoted.Sample collection method has two kinds, the selectivity sample collecting (Part Sample Collection, PSC) and whole sample collecting (WholeSample Collection, WSC).

PSC promptly scrapes faecal samples top layer mucous membrane with aseptic disposable cutter, divides to install in the 2ml centrifuge tube the about 200mg sample of every pipe.Be applicable to the collection of large mammal fresh excreta.WSC puts into 50ml-100ml centrifuge tube with whole ight soil, determines its weight.

Because endonuclease is considered to the principal element of degradation of dna, so sample must be preserved after collection as early as possible.Most of store methods all are that faecal samples is carried out low temperature, dehydration or it is kept at preserving in the liquid to reduce endonuclease activity.Subzero treatment is a cold method, general-20 ℃; Processed is a desiccating method, and silica dehydrator, silicon-dioxide drying, microwave drying etc. are arranged; Preserve liquid commonly used dehydrated alcohol, 70% ethanol, methyl-sulphoxide (DMSO), methyl-sulphoxide saturated salt solution (DETs) etc. are arranged.

The different method of samplings and store method are adapted to different plant species and site conditions uses down.

Sample with the collection of PSC method can directly carry out DNA extraction to sample.

With carrying out secondary sample before the sample extraction of WSC method collection, promptly from ight soil, get and be rich in cell and impurity carries out DNA extraction than small part.At present commonly used mainly contain 4 kinds of secondary sample methods: 1. buffer solution elution ight soil superficial cell; 2. scrape the ight soil surface; 3. top layer sampling; 4. homogeneous sampling.

Different secondary sample methods is adapted to the sampling of different store method samples.

Can the key of studying for the material extraction animal DNA with ight soil be extract highly purified faeces DNA.After the faeces DNA analytical technology is risen, made huge achievement in this respect both at home and abroad, developed multiple faeces DNA extracting method (Constable etal., 1995; Savill et al., 2001; Zhong Hua etc., 2003; Zhang Baowei etc., 2004), fully confirmed the feasibility (Fernando et al., 2003) that faeces DNA is analyzed.But still there is following problem in the practical application:

1, various extracting method versatilities are bad; In the different experiments case, there is the difference of amplification success rate between species and individuality.This is to hinder the major obstacle that the faeces DNA extractive technique is promoted always.

2, reckon without the influence of relevant link with DNA extraction to the result; Because the singularity of fecal material, its extraction result is subjected to the influence of multiple factor, as sample collecting, preservation (Frantzen et al., 1998; Piggott and Taylor, 2003) or the like, ignore wherein any one link, all can bring disadvantageous effect even cause test failure to test-results.The domestic and foreign literature data is not all introduced these influence factors at present, when therefore carrying out faeces DNA extraction according to the method that provides in having published thesis abroad, often can not get expected effect.Meanwhile,, both can't from existing documents and materials, obtain enough information and instruct faeces DNA to extract test, also can't from failure, analyze reason for many scholars that just begun the research of molecule scatology.

Three, summary of the invention

The present invention is directed to the experimental study that above-mentioned existing in prior technology weak point is carried out, aim to provide the extraction method of mammal faeces DNA that a kind of versatility is good, reliability is high, with low cost, advance the popularization of faeces DNA technology.

The extraction method of mammal faeces DNA that the present invention is alleged its essence is the DNA that extracts in the defecation mammalian cell in ight soil.Technical problem to be solved is that DNA is discharged in cell, and is not destroyed in a series of separation, extraction and purge process, thereby obtains the higher mammalian DNA sample of purity.

This extracting method comprises feces collection, preserve, sampling, pre-treatment and DNA extraction, it is characterized in that: described DNA extraction is to separate through cracking in sample, separate out with purification process after obtain DNA, it promptly is to add in sample by Tris-HCl that described cracking separates, the lysis buffer that EDTA and cetyl trimethylammonium bromide (CTAB) are mixed with, under 50～60 ℃ of conditions, leave standstill 1.5～2.5h, centrifugation, get supernatant liquor, going out described the separating out of DNA with the mixed organic solvents extracting is to leave standstill 0.5～1h add the abundant mixing of dehydrated alcohol of the NaAc of 2～4M concentration and 1～2 times of volume in the extract that contains DNA after under-10～-30 ℃ of conditions, centrifugation is with dry under the DNA precipitation room temperature of separating out; Described purifying is that the Tris-HCl damping fluid and guanidine thiocyanate (GuSCN) to the precipitation that add pH6～7 in precipitation are dissolved fully, add silicon-dioxide microballon suspension then, shake up, centrifugation, precipitate with 70% washing with alcohol silica bead, separate, add TE (Tris-edta buffer liquid) liquid wash-out after sloughing ethanol remaining in the silica bead, centrifugation obtains containing the TE liquid of DNA;

Described mixed organic solvents is that phenol, chloroform and primary isoamyl alcohol mix the mixed solvent that obtains by the volume ratio of 25:24:1.

Sample is carried out pre-treatment be intended to remove preservation liquid remaining in the sample.Therefore the sample of taking from non-preservation liquid preservation then be need not to carry out pre-treatment.Pretreated method is used physiological saline immersion, the washing sample of phosphate buffered saline buffer (PBS) or 0.9% exactly, to remove remaining preservation liquid.

CTAB is a kind of cationic surfactant, uses CTAB cracking zooblast, released dna in the present invention, and the while separates with DNA with other plurality of impurities formation precipitations in the ight soil.

GuSCN has the effect that makes the nucleic acid hydrolysis enzyme deactivation, but the existence of GuSCN makes silicon-dioxide (SiO in the present invention ₂) effect of specific adsorption DNA arranged, with separate, purify DNA.

The product D NA purity that present method is extracted is greater than 1.7 (OD ₂₆₀/ OD ₂₈₀), DNA product segment can be carried out gene amplification and order-checking greater than 10Kb.Present method is all effective with interior sample to preserving 3 years.

Present method has good versatility, and the faeces DNA that is widely used in various feeding habits animals extracts, and does not generally need repeated experiments.10 kinds of animals (macaque (Macaca mulatta) in checking, black muntjac (Muntiacus crinifrons), spotted deer (Cervusnippon), giraffe (Giraffa camelopardalis), red deer (Cervus elaphus), yak (Bosgrunniens), Squirrel monkey (Saimiri sciureus), porcupine (Hystrix brachyurus), during arctic fox (Alopex lagopus) and racoon dog (Nyctereutesprocy) and faeces DNA extract, only once extract and promptly obtain high-quality template DNA, in the amplification to the 12SrRNA gene, the faeces DNA template of all extractions all can successfully increase.Obtain and the 450bp product of estimating that size is identical, and concentration is big, high specificity, sequencing result has confirmed the verity of extracting.

Four, description of drawings

Fig. 1, from the extracting of stump-tailed macaque ight soil to dna profiling electrophoresis detection figure.

Can extract high-quality faeces DNA with present method from ight soil, not only concentration is big, and less degradation.

The concentration gradient PCR result of Fig. 2 stump-tailed macaque 12S rRNA gene, template are the faeces DNA that modified version CTAB method is extracted.Template concentrations is followed successively by 10 ^-4, 10 ^-3, 10 ^-2, 10 ^-1, 1,5,10,15,20 units (1 unit=1 μ l template DNA).M is the DNAladder of 100bp.

The concentration gradient PCR result of Fig. 3 stump-tailed macaque 12S rRNA gene, template are the faeces DNA that dedicated kit extracts.Template concentrations is followed successively by 10 ^-4, 10 ^-3, 10 ^-2, 10 ^-1, 1,5,10,15,20 units (1 unit=1 μ l template DNA).M is the DNA ladder of 100bp.

Because pcr amplification needs certain density template just can carry out smoothly, therefore can compare the concentration that two kinds of methods extract product by the test of template dilution PCR gradient. Increase progressively in the test in template concentrations, owing to the increase of mortifier concentration contained in the PCR system along with the template amount increases, finally can reach the concentration that suppresses the Taq enzymatic activity, therefore relatively can carry out smoothly the critical concentration of each template of pcr amplification, then can compare the purity of two kinds of products that method is extracted. As shown in Figures 2 and 3, increase progressively in the test in template concentrations, the template that this patent method extracts can add to 15 units and not affect normally carrying out of amplification, and the template that kit extracts can only add to 10 units, when being added into 15 unit templates, then can normally carry out because mortifier amount in the PCR reaction system too much hinders pcr amplification. Therefore can reach a conclusion: the faeces DNA that this method extracts is compared with professional kit, although production concentration is suitable, purity is higher than professional kit. This method is kind of a high-purity faeces DNA extracting method.

Five, embodiment

Be example with the stump-tailed macaque now, non-limiting examples is described below:

1, feces collection: gather the faecal samples that stump-tailed macaque defecation, exposure in the open air is no more than 24 hours;

Adopt the selectivity sample collecting, scrape faecal samples top layer mucous membrane, divide to install in the 2ml centrifuge tube every pipe 180-220mg sample with aseptic disposable blade; In addition adopt whole sample collection, whole ight soil sample is put into 50ml-100ml centrifuge tube, determines its weight;

2, sample retention: preserve after the method that sample is preserved comprises cryopreservation or processed, or in the high salt concentration damping fluid, preserve; Described cryopreservation adopts cold method, freezing temp-20 ℃～-70 ℃; Described processed is to adopt dehydrated alcohol dehydration or silicon-dioxide kept dry; It is to adopt the saturated salt solution (DETs) of methyl-sulphoxide to preserve sample that described high salt is handled.

3, sampling reaches and pre-treatment:

Directly carry out DNA extraction for the sample of being preserved, or carry out DNA extraction again by secondary sample; Described secondary sample is one of following four kinds of modes: with buffer solution elution ight soil superficial cell; Scrape and get the ight soil surface; The top layer sampling; The homogeneous sampling;

Described pre-treatment is the preservation liquid of removing in the sample;

4, DNA extraction:

Experiment is prepared:

Be ready to range and be respectively the micropipet of 1ml, 200 μ l, 50 μ l; The water-bath temperature transfers to 55 ℃; Supercentrifuge centrifugal force is wanted to reach 10000g; Guarantee that 1.5ml and 2ml centrifuge tube all sterilize; Guarantee that all solution do not have precipitation, no crystallization, if precipitation or crystallization are arranged, available 70 ℃ of water-baths allow its dissolving; Dehydrated alcohol is placed-20 ℃ freezing; TE is heated to 65 ℃ of dissolving or wash-outs that are beneficial to DNA.

Testing sequence:

1. get 200mg ight soil in the 2ml centrifuge tube, add in the 1ml CTAB lysate, 55 ℃ of water-baths are 2 hours behind the mixing, take out every half an hour to shake up once; Described lysate is to add 1～3wt%CATB, 15～40m mol/L EDTA and 1～2mol/L NaCl among pH7.5～8.5, the 80～200m mol/L Tris-HCl;

2. the centrifugal 5min of 12000rpm shifts out after the centrifugal end in the aseptic 1.5ml centrifuge tube of 700 μ l supernatants to, abandons precipitation.In supernatant, add equal-volume phenol: chloroform: primary isoamyl alcohol (25:24:1), the centrifugal 10min of 10000rpm behind the concussion 10min; The sucking-off supernatant adds equal-volume phenol: chloroform to another aseptic 1.5ml centrifuge tube: primary isoamyl alcohol (25:24:1) extracting again no longer includes white precipitate between centrifugal back water and organic phase;

3. the 3M NaAc that adds 0.1 times of volume in supernatant, the dehydrated alcohol of 1.5 times of volumes of adding (adding the volume that calculates after the salt) precooling behind the mixing is placed 0.5～1h for-20 ℃ behind the abundant mixing on vortex mixer.

4. the centrifugal 10min of 12000rpm inhales and abandons supernatant, precipitates airing at room temperature;

5. in precipitation, add 1ml GuSCN washing lotion, shake up to precipitation dissolving fully; Described washing lotion is to add 5～8mol/L GuSCN among pH6～7, the 80～200m mol/L Tris-HCl;

6. add 100 μ l silica bead suspensions, slowly shake up adsorption of DNA 10min, the centrifugal 5min of 10000rpm inhales and abandons supernatant reservation silica bead precipitation;

7. distinguish washing precipitation 2 times with 500 μ l GuSCN washing lotions and 500 μ l70% ethanol;

8. the centrifugal supernatant of abandoning, the 1.5ml centrifuge tube is placed 5min oven dry residual ethanol in 55 ℃ of baking ovens, in the silica bead precipitation, add 100 μ lTE again, concussion mixing 5min eluted dna, the last centrifugal 5min of 12000rpm shifts out in aseptic 200 μ l of about 80 μ l supernatants to one or the 1.5ml centrifuge tube and preserves.

Claims (2)

1. extraction method of mammal faeces DNA, comprise feces collection, preservation, sampling, pre-treatment and DNA extraction, it is characterized in that: described DNA extraction be in sample through cracking separate, separate out with purification process after obtain DNA, it promptly is to add the lysis buffer that is mixed with by Tris-HCl, EDTA and cetyl trimethylammonium bromide in sample that described cracking separates, under 50～60 ℃ of conditions, leave standstill 1.5～2.5h, centrifugation, get supernatant liquor, go out DNA with the mixed organic solvents extracting; Described separating out is to leave standstill 0.5～1h add the abundant mixing of dehydrated alcohol of the NaAc of 2～4M concentration and 1～2 times of volume in the extract that contains DNA after under-10～-30 ℃ of conditions, and centrifugation is with dry under the DNA precipitation room temperature of separating out; Described purifying is that the Tris-HCl damping fluid and guanidine thiocyanate to the precipitation that add pH6～7 in precipitation are dissolved fully, add silicon-dioxide microballon suspension then, shake up, centrifugation, precipitate with 70% washing with alcohol silica bead, separate, add TE liquid wash-out after sloughing ethanol remaining in the silica bead, centrifugation obtains containing the TE liquid of DNA;

Described mixed organic solvents is that phenol, chloroform and primary isoamyl alcohol mix the mixed solvent that obtains by the volume ratio of 25:24:1.

2. extracting method according to claim 1 is characterized in that: described pre-treatment is exactly physiological saline immersion, the washing sample with phosphate buffered saline buffer or 0.9%.

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