CN102864138B - Human feces DNA extraction method - Google Patents
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Abstract
The invention discloses a human feces DNA extraction method. The prior art is complicated and time-consuming; and the trace DNAs extracted by the previous sampling method is too little to perform molecular diagnosis. The extraction method uses selective sample collection PSC equipment for feces samples and adopts a low temperature preservation method. The extraction method comprises the following steps: picking a sample by using a sterilized gun head or sterilized blade and directly placing the picked sample on ice; and adding a feces lysis solution, fully cracking and evenly mixing, adding bovine serum albumin, centrifuging to remove the supernatant, adding protease K to perform a digestion reaction at 58 DEG C, then adding a protein precipitation solution to precipitate the protein, centrifuging to obtain the supernatant, adding a silica gel membrane binding solution to fully bind DNAs, centrifuging to obtain precipitates, adding a silica gel membrane washing solution to wash twice, centrifuging, and adding a TE (Tris + EDTA) buffer solution to finally obtain a preservation solution with DNAs. The human feces DNA extraction method has the advantages of high repeatability and stability, simpleness and short time.
Description
Technical field
The invention belongs to biological technical field, relate to the method for extracting DNA in non-damage sampling material, be specifically related to a kind of human feces DNA extraction method.
Background technology
Colorectal cancer is modal malignant tumor of digestive tract, and incidence is only second to cancer of the stomach and esophagus cancer.The sickness rate that recent two decades carrys out colorectal cancer is increasing gradually, meanwhile, and its age of onset trend aging.At western developed country, colorectal cancer is the second malignant neoplasm that is only second to lung cancer.Some researchs in recent years show K-ras, P53, APC, the sudden change of some genes such as MMR can cause the cell transformation of Normal Colon mucosal epithelium to become tumour cell (Vogelstein, B. and Kinzler, K.W., 1993), to the progress of these transgenations, making to detect Colorectal Cancer Diagnosis by faeces DNA becomes a kind of possibility.Normal people's ight soil approximately can be drained and approach 10 every day
10individual epithelial cell, carries out to the colonic mucosa cell being shed in ight soil a kind of desirable examination means that Molecular Detection is colorectal cancer.Research field (Parsons, the B of a focus have been become in recent years about the Disease-causing gene of attempting to detect colorectal cancer from faecal samples
et al., 1995; Sidransky, D
et al., 1992; Ahlquist, D.A
et al., 2000; Dong, S.M
et al., 2001).But before carrying out clinical diagnosis by this method, also have many difficult problems to need to solve, such as, in the collection of sample and sepn process, tend to because the degraded of DNA enzyme, the existence of various enzyme co-inhibitors (mucus, bacterium and swill etc.) and cholate causes the human DNA output DNA considerably less or that extract extracting from ight soil to be difficult to use in pcr amplification.For the reliable and stable molecular diagnosis that carries out colorectal cancer, also there is in recent years many research, contrast to improve quality (Monteiro, the L of DNA sample by many groups of purifications and amplification method
et al., 2001; Deuter, R
et al., 2001; Machiels, B.M
et al., 2000; Ito, Y
et al., 2002).But all very loaded down with trivial details and consuming time (such as application of a large amount of phenol) of most research method operation, more existing faeces DNAs extract test kit and can not solve these difficult problems completely on the market, and expensive.
In recent years, gene diagnosis examine in the morning of mankind's kinds of tumors, disease, in widespread attention in treatment and prognosis evaluation.The primary work of carrying out gene diagnosis is the collection of sample and the extraction of DNA sample.The past method of sampling mainly contains two kinds: the one, and nocuity samples, and obtains patient's the tissue samples such as pathological tissue and blood, and this method can cause pain to patient; The 2nd, nondestructive sampling, by obtaining patient's the analytic sample such as hair, nail, although the method can not cause pain to patient, the trace amount DNA extracting from these samples due to content very little, be difficult to for carrying out molecular diagnosis.
Non-damage sampling is in the situation that not touching or do not injure human body itself, and the multi-form analytic samples such as hair by collector, ight soil, urine, swill (containing the cell that oral cavity comes off) carry out extraction and the analysis of DNA.In non-damage sampling material, ight soil is a kind of desirable analytic sample, and reason comprises: one, mankind's defecation has rhythmicity, and with respect to other non-damages sampling materials, sample abundance, is easy to gather; Two, faecal samples can directly obtain in the situation that not contacting sample source people, little to people's interference; Three, gather fecal sample and can significantly reduce sampling cost cost.Therefore the method has been got rid of all unfavorable factor of tradition to patient's sampling method, successfully applied in recent years species and differentiated that (by the amplification to Mitochondrial DNA), individual recognition (micro-satellite amplification), Animal Sex identify (sry gene on Y chromosome is increased), plant the evaluation (plastosome control D ring is increased), maternal evaluation (by the amplification to Mitochondrial DNA), paternity test (micro-satellite is increased), Estimating population size (micro-satellite is increased) of edge relation etc. (Kohn and Wayne, 1997).These researchs have promoted the progress of the sampling method before collection and store method and the extraction of faecal samples.
Owing to containing a lot of bacteriums in ight soil, endonuclease, sample DNA is easy to degraded, need preserve as early as possible so gather the faecal samples coming, and store method is generally subzero treatment, i.e. freezing preservation.
Can the key of studying as material extraction DNA take ight soil be extract highly purified DNA.After Fecal DNA analysis grows up, obtain in this respect huge progress both at home and abroad, develop multiple faeces DNA extracting method (Constable
et al., 1995; Savill
et al., 2001; Zhong Hua etc., 2003; Zhang Baowei etc., 2004).But in practical application, still there is a difficult problem: due to the singularity of fecal material, as the collection of sample, preserve etc. related link factor and all can have influence on the result (Frantzen of DNA extraction
et al., 1998; Piggott and Taylor, 2003).Domestic and foreign literature data is not all introduced these influence factors at present, therefore carry out faeces DNA extraction according to the method providing in the paper of having delivered both at home and abroad, cannot from existing documents and materials, obtain enough information and instruct faeces DNA to extract research, can not get expected effect.
Summary of the invention
The present invention is directed to the experimental study that the existing weak point of above-mentioned prior art is carried out, object, people's the faeces DNA extracting method of a kind of fast simple, low cost, high reliability is provided, advances people's faeces DNA technology popularization.
A kind of human feces DNA extraction method, its essence is the DNA in the cell that extracts defecation people in ight soil, technical problem to be solved is that DNA is discharged in cell, and not being destroyed in a series of separation, precipitation and purge process, thereby the higher people's of the concentration of obtaining and purity DNA sample.
The present invention is for solving its technical problem, and the method steps of taking is as follows:
Step 1. the collection of faecal samples
The exposure that gathers people's discharge is no more than the faecal samples of 24 hours, adopts selectivity sample collecting PSC, scrapes the top layer mucous membrane of faecal samples by sterile razor blade, divides and installs in 2ml centrifuge tube;
Step 2. the preservation of faecal samples
Adopt Cryopreservation of Viable, gather the faecal samples of coming, be put in immediately-20 ℃～-80 ℃ preservations;
Step 3. the sampling of faecal samples
For preserved faecal samples, take out and be placed on ice, with the sample of sterilizing rifle head or sterilizing blade picking 180～200mg, be placed on ice;
Step 4. the DNA extraction of sample
(1). lysis separates
In sample, add the ight soil lysate of 1.2～1.6ml, be placed in abundant cracking under normal temperature condition and mix rear centrifugation, get supernatant liquor, add 200～300 mg bovine serum albumin BSA to remove impurity, centrifugation obtains supernatant liquor, the Proteinase K that the concentration that adds 20～40 μ l in this supernatant liquor is 20mg/ml is placed in digestion reaction 1～6h under 58 ℃ of conditions, obtains Digestive system;
Described ight soil lysate is formulated by 50mmol/L Tris-Cl, 5mmol/L EDTA, 1mmol/L NaCl and 0.1% (W/V) sodium lauryl sulphate SDS;
(2). precipitation
The Digestive system ice bath of above-mentioned steps (1) gained is cooled to normal temperature, then adds 200～400 μ l albumen precipitation liquid precipitate albumen, after fully mixing, remove albumen impurity through centrifugation, obtain the supernatant liquor after centrifugal, this supernatant liquor is DNA extract;
(3). purifying
Add pellosil in conjunction with liquid to removing in the DNA extract of albumen impurity, fully in conjunction with DNA, DNA extract is combined the volume ratio of liquid with pellosil be 1:(1.5～2.5), then adsorb through pellosil, centrifugation, be precipitated thing, in throw out, add 700～800 μ l pellosil washings washings, twice of repeated washing, then centrifugation is sloughed after pellosil washings remaining in pellosil, add 50～100 μ lTE damping fluids to dissolve eluted dnas, centrifugation obtains the supernatant liquor after centrifugal, and this supernatant liquor is the preservation liquid containing DNA;
Described pellosil is 3mol/L Guanidinium hydrochloride in conjunction with liquid;
The ethanol that described pellosil washings is 50% by 2mmol/L Tris-Cl, 1mmol/L EDTA, 5mmol/L NaCl and volume content is formulated;
Described albumen precipitation liquid is that 11.5% glacial acetic acid is formulated by 2mol/L potassium acetate and volume content.
Illustrate further, the extraction of DNA is without soaking, wash these preprocessing process through phosphate buffered saline buffer or physiological saline, and the fecal sample that can directly get rapidly 180～200mg people is placed on ice, carries out DNA extraction.
The DNA fragmentation length that the present invention obtains is greater than 20Kb, and OD260/OD280 ratio is greater than 1.7, can be directly used in PCR and other molecular biology purposes.
Faecal samples is placed in rapidly operation on ice and is intended to prevent DNA degradation.In the fecal sample of first cracking, add BSA to be intended to the co-inhibitors such as the cholate that contains in abundant Adsorption ight soil.
The beneficial effect that the present invention has:
The present invention has higher repetition stability, do not need to repeat experiment, in 50 routine volunteer's faeces DNAs extract, only once extract and obtain high-quality template DNA, in to K-ras the 12nd, 13 amino acid whose amplifications, the faeces DNA template of all extractions all can successfully increase, and obtains and the product of estimating that clip size is identical, and sequencing result has also confirmed reliability of the present invention.
Embodiment
The present invention is described further below.
Now, take normal people's ight soil as example, non-limiting examples is described below:
Embodiment 1
1. faecal samples collection:
The exposure that gathers people's discharge is no more than the faecal samples of 24 hours; Adopt selectivity sample collecting PSC, scrape the top layer mucous membrane of faecal samples by sterile razor blade, divide and install in 2ml centrifuge tube;
2. sample retention:
Adopt Cryopreservation of Viable, gather the faecal samples of coming, be put in immediately-20 ℃ of preservations;
3. sampling:
For preserved faecal samples, take out and be placed on ice, with the sample of sterilizing rifle head or sterilizing blade picking 180mg, be placed on ice;
4.DNA extracts:
Extraction step:
(1) get ight soil lysate 1.2ml and join in the sterilized Eppendorf pipe of 2ml, and place operation on ice;
(2) fecal sample that takes 180mg is in each Eppendorf pipe, until the abundant cracking of sample mixes;
(3) the centrifugal 5min of maximum speed at normal temperatures;
(4) get supernatant liquor in new 2ml Eppendorf pipe, repeated centrifugation once, is removed the broken particulate matter in fecal sample, by supernatant liquor to new 2ml Eppendorf pipe;
(5) take 200mg BSA and join in each sample, whirlpool mixes until BSA suspends completely rapidly, hatches suspended substance 5min under normal temperature, and its some supressors are absorbed by BSA;
(6) high speed centrifugation 5min at normal temperatures, removes excrement particles and is attached to the supressor on BSA;
(7) centrifugal end, takes out rapidly supernatant liquor to new 2ml Eppendorf pipe, abandons throw out;
(8) high speed centrifugation 5min again, gets supernatant liquor to new 2ml Eppendorf pipe;
(9) adding the concentration of 20 μ l is 20mg/ml Proteinase K, 58 ℃ of water-bath digestion reaction 1h;
(10) sample ice bath 5min is cooled to normal temperature, adds 200 μ l albumen precipitation liquid, whirlpool acutely mixes 20s, again ice bath 5min;
(11) high speed centrifugation 5min, protein precipitation;
(12) getting supernatant liquor is added in new 2ml Eppendorf pipe;
(13) in supernatant liquor, add DNA pellosil in conjunction with liquid, supernatant liquor is combined the volume ratio of liquid with DNA pellosil be 1:1.5, and whirlpool mixes 15s;
(14) take out 600ml mixture and join in the purification column in 2ml Eppendorf pipe, under normal temperature, hatch 5min, cover lid, the centrifugal 1min of 5000rpm/min, discards filtrate;
(15) repeating step (14) twice, makes all lysates all filter the pellosil of purification column bottom;
(16) silicagel column is placed in new 2ml Eppendorf pipe, discards the Eppendorf pipe that contains filtrate;
(17) open gently purification column lid, add 700 μ l pellosil washingss, cover lid, the centrifugal 1min of 5000rpm/min, discards the Eppendorf pipe that contains filtrate;
(18) use pellosil washings repeated washing once, purification column is placed in new 2ml Eppendorf pipe to the centrifugal 1min of maximum speed;
(19) purification column is put in new 1.5ml Eppendorf pipe, the TE damping fluid of getting 50 μ l is directly added on pellosil, and cover lid, reacts 5min under normal temperature, high speed centrifugation 1min, the DNA that obtains washing from purification column;
(20) from 1.5ml Eppendorf pipe, take out gently the filtrate of washing, rejoin on purification column, react 5min under normal temperature, high speed centrifugation once obtains DNA and preserves liquid again;
(21) DNA is preserved to liquid in-20 ℃ of preservations.
Embodiment 2
1. faecal samples collection:
The exposure that gathers people's discharge is no more than the faecal samples of 24 hours; Adopt selectivity sample collecting PSC, scrape the top layer mucous membrane of faecal samples by sterile razor blade, divide and install in 2ml centrifuge tube;
2. sample retention:
Adopt Cryopreservation of Viable, gather the faecal samples of coming, be put in immediately-80 ℃ of preservations;
3. sampling:
For preserved faecal samples, take out and be placed on ice, with the sample of sterilizing rifle head or sterilizing blade picking 200mg, be placed on ice;
4.DNA extracts:
Extraction step:
(1) get ight soil lysate 1.6ml and join in the sterilized Eppendorf pipe of 2ml, and place operation on ice;
(2) fecal sample that takes 200mg is in each Eppendorf pipe, until the abundant cracking of sample mixes;
(3) the centrifugal 5min of maximum speed at normal temperatures;
(4) get supernatant liquor in new 2ml Eppendorf pipe, repeated centrifugation once, is removed the broken particulate matter in fecal sample, by supernatant liquor to new 2ml Eppendorf pipe;
(5) take 300mg BSA and join in each sample, whirlpool mixes until BSA suspends completely rapidly, hatches suspended substance 5min under normal temperature, and its some supressors are absorbed by BSA;
(6) high speed centrifugation 5min at normal temperatures, removes excrement particles and is attached to the supressor on BSA;
(7) centrifugal end, takes out rapidly supernatant liquor to new 2ml Eppendorf pipe, abandons throw out;
(8) high speed centrifugation 5min again, gets supernatant liquor to new 2ml Eppendorf pipe;
(9) adding the concentration of 40 μ l is 20mg/ml Proteinase K, 58 ℃ of water-bath digestion reaction 6h;
(10) sample ice bath 5min is cooled to normal temperature, adds 400 μ l albumen precipitation liquid, whirlpool acutely mixes 20s, again ice bath 5min;
(11) high speed centrifugation 5min, protein precipitation;
(12) getting supernatant liquor is added in new 2ml Eppendorf pipe;
(13) in supernatant liquor, add DNA pellosil in conjunction with liquid, supernatant liquor is combined the volume ratio of liquid with DNA pellosil be 1:2.5, and whirlpool mixes 15s;
(14) take out 600ml mixture and join in the purification column in 2ml Eppendorf pipe, under normal temperature, hatch 5min, cover lid, the centrifugal 1min of 5000rpm/min, discards filtrate;
(15) repeating step (14) twice, makes all lysates all filter the pellosil of purification column bottom;
(16) silicagel column is placed in new 2ml Eppendorf pipe, discards the Eppendorf pipe that contains filtrate;
(17) open gently purification column lid, add 800 μ l pellosil washingss, cover lid, the centrifugal 1min of 5000rpm/min, discards the Eppendorf pipe that contains filtrate;
(18) use pellosil washings repeated washing once, purification column is placed in new 2ml Eppendorf pipe to the centrifugal 1min of maximum speed;
(19) purification column is put in new 1.5ml Eppendorf pipe, the TE damping fluid of getting 100 μ l is directly added on pellosil, and cover lid, reacts 5min under normal temperature, high speed centrifugation 1min, the DNA that obtains washing from purification column;
(20) from 1.5ml Eppendorf pipe, take out gently the filtrate of washing, rejoin on purification column, react 5min under normal temperature, high speed centrifugation once obtains DNA and preserves liquid again;
(21) DNA is preserved to liquid in-20 ℃ of preservations.
Embodiment 3
1. faecal samples collection:
The exposure that gathers people's discharge is no more than the faecal samples of 24 hours; Adopt selectivity sample collecting PSC, scrape the top layer mucous membrane of faecal samples by sterile razor blade, divide and install in 2ml centrifuge tube;
2. sample retention:
Adopt Cryopreservation of Viable, gather the faecal samples of coming, be put in immediately-50 ℃ of preservations;
3. sampling:
For preserved faecal samples, take out and be placed on ice, with the sample of sterilizing rifle head or sterilizing blade picking 190mg, be placed on ice;
4.DNA extracts:
Extraction step:
(1) get ight soil lysate 1.4ml and join in the sterilized Eppendorf pipe of 2ml, and place operation on ice;
(2) fecal sample that takes 190mg is in each Eppendorf pipe, until the abundant cracking of sample mixes;
(3) the centrifugal 5min of maximum speed at normal temperatures;
(4) get supernatant liquor in new 2ml Eppendorf pipe, repeated centrifugation once, is removed the broken particulate matter in fecal sample, by supernatant liquor to new 2ml Eppendorf pipe;
(5) take 250mg BSA and join in each sample, whirlpool mixes until BSA suspends completely rapidly, hatches suspended substance 5min under normal temperature, and its some supressors are absorbed by BSA;
(6) high speed centrifugation 5min at normal temperatures, removes excrement particles and is attached to the supressor on BSA;
(7) centrifugal end, takes out rapidly supernatant liquor to new 2ml Eppendorf pipe, abandons throw out;
(8) high speed centrifugation 5min again, gets supernatant liquor to new 2ml Eppendorf pipe;
(9) adding the concentration of 30 μ l is 20mg/ml Proteinase K, 58 ℃ of water-bath digestion reaction 4h;
(10) sample ice bath 5min is cooled to normal temperature, adds 300 μ l albumen precipitation liquid, whirlpool acutely mixes 20s, again ice bath 5min;
(11) high speed centrifugation 5min, protein precipitation;
(12) getting supernatant liquor is added in new 2ml Eppendorf pipe;
(13) in supernatant liquor, add DNA pellosil in conjunction with liquid, supernatant liquor is combined the volume ratio of liquid with DNA pellosil be 1:2, and whirlpool mixes 15s;
(14) take out 600ml mixture and join in the purification column in 2ml Eppendorf pipe, under normal temperature, hatch 5min, cover lid, the centrifugal 1min of 5000rpm/min, discards filtrate;
(15) repeating step (14) twice, makes all lysates all filter the pellosil of purification column bottom;
(16) silicagel column is placed in new 2ml Eppendorf pipe, discards the Eppendorf pipe that contains filtrate;
(17) open gently purification column lid, add 750 μ l pellosil washingss, cover lid, the centrifugal 1min of 5000rpm/min, discards the Eppendorf pipe that contains filtrate;
(18) use pellosil washings repeated washing once, purification column is placed in new 2ml Eppendorf pipe to the centrifugal 1min of maximum speed;
(19) purification column is put in new 1.5ml Eppendorf pipe, the TE damping fluid of getting 75 μ l is directly added on pellosil, and cover lid, reacts 5min under normal temperature, high speed centrifugation 1min, the DNA that obtains washing from purification column;
(20) from 1.5ml Eppendorf pipe, take out gently the filtrate of washing, rejoin on purification column, react 5min under normal temperature, high speed centrifugation once obtains DNA and preserves liquid again;
(21) DNA is preserved to liquid in-20 ℃ of preservations.
Above-mentioned pellosil is 3mol/L Guanidinium hydrochloride in conjunction with liquid; Pellosil washings is that 2mmol/L Tris-Cl, 1mmol/L EDTA, 5mmol/L NaCl and volume content are 50% ethanol is formulated; Albumen precipitation liquid is to be the formulated mixed solution of 11.5% glacial acetic acid by 2mol/L potassium acetate and volume content.
Above-mentioned ight soil lysate is formulated by 50mmol/L Tris-Cl, 5mmol/L EDTA, 1mmol/L NaCl and 0.1% (W/V) sodium lauryl sulphate SDS; Pellosil is 3mol/L Guanidinium hydrochloride in conjunction with liquid; The ethanol that pellosil washings is 50% by 2mmol/L Tris-Cl, 1mmol/L EDTA, 5mmol/L NaCl and volume content is formulated; Albumen precipitation liquid is that 11.5% glacial acetic acid is formulated by 2mol/L potassium acetate and volume content.
Claims (2)
1. a human feces DNA extraction method, is characterized in that the method comprises the following steps:
Step 1. the collection of faecal samples
The exposure that gathers people's discharge is no more than the faecal samples of 24 hours, adopts selectivity sample collecting, scrapes the top layer mucous membrane of faecal samples by sterile razor blade, divides and installs in 2ml centrifuge tube;
Step 2. the preservation of faecal samples
Adopt Cryopreservation of Viable, gather the faecal samples of coming, be put in immediately-20 ℃～-80 ℃ preservations;
Step 3. the sampling of faecal samples
For preserved faecal samples, take out and be placed on ice, with the sample of sterilizing rifle head or sterilizing blade picking 180～200mg, be placed on ice;
Step 4. the DNA extraction of sample
(1). lysis separates
In sample, add the ight soil lysate of 1.2～1.6ml, be placed in abundant cracking under normal temperature condition and mix rear centrifugation, get supernatant liquor, add 200～300mg bovine serum albumin BSA to remove impurity, centrifugation obtains supernatant liquor, the Proteinase K that the concentration that adds 20～40 μ l in this supernatant liquor is 20mg/ml is placed in digestion reaction 1～6h under 58 ℃ of conditions, obtains Digestive system;
Described ight soil lysate is that 0.1% sodium lauryl sulphate SDS is formulated by 50mmol/L Tris-Cl, 5mmol/L EDTA, 1mmol/LNaCl and weightmeasurement ratio;
(2). precipitation
The Digestive system ice bath of above-mentioned steps (1) gained is cooled to normal temperature, then adds 200～400 μ l albumen precipitation liquid precipitate albumen, after fully mixing, remove albumen impurity through centrifugation, obtain the supernatant liquor after centrifugal, this supernatant liquor is DNA extract;
(3). purifying
Add pellosil in conjunction with liquid to removing in the DNA extract of albumen impurity, DNA extract is combined the volume ratio of liquid with pellosil be 1:1.5～1:2.5, then adsorb through pellosil, centrifugation, be precipitated thing, in throw out, add 700～800 μ l pellosil washings washings, twice of repeated washing, then centrifugation is sloughed after pellosil washings remaining in pellosil, add 50～100 μ l TE damping fluids to dissolve eluted dna, centrifugation obtains the supernatant liquor after centrifugal, and this supernatant liquor is the preservation liquid containing DNA;
Described pellosil is 3mol/L Guanidinium hydrochloride in conjunction with liquid;
The ethanol that described pellosil washings is 50% by 2mmol/L Tris-Cl, 1mmol/L EDTA, 5mmol/LNaCl and volume content is formulated;
Described albumen precipitation liquid is that 11.5% glacial acetic acid is formulated by 2mol/L potassium acetate and volume content.
2. a kind of human feces DNA extraction method according to claim 1, it is characterized in that: the extraction of DNA does not comprise that phosphate buffered saline buffer or physiological saline soak, washing preprocessing process, the fecal sample of directly getting rapidly 180～200mg people is placed on ice, carries out DNA extraction.
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