CN104328108A - Method of quickly extracting and purifying DNA from formalin-fixed tissue - Google Patents
Method of quickly extracting and purifying DNA from formalin-fixed tissue Download PDFInfo
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- CN104328108A CN104328108A CN201410383891.3A CN201410383891A CN104328108A CN 104328108 A CN104328108 A CN 104328108A CN 201410383891 A CN201410383891 A CN 201410383891A CN 104328108 A CN104328108 A CN 104328108A
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Abstract
The invention discloses a method of quickly extracting and purifying DNA from formalin-fixed tissue and belongs to the technical field of forensic medicine. The method includes following steps: (1) washing the formalin-fixed tissue with distilled water, breaking the formalin-fixed tissue and drying the tissue with filter papers; (2) soaking the formalin-fixed tissue in anhydrous ethanol for 1-5 min, performing centrifugation with a supernate being abandoned, and then successively adding ethanol with the concentration being 70-80% and ethanol with concentration being 40-60% and repeating the steps hereinbefore with a precipitation being retained; (3) adding a cell lysis buffer, sodium dodecyl sulfate and a protease K, performing incubation at 55-65 DEG C, performing centrifugation with a supernate for later use; (4) adding salt to the supernate with mixing uniformly, allowing the supernate for stand at -4 - 4 DEG C for layering, performing centrifugation with the supernate for later use; and (5) adding a DNA binding solution with mixing uniformly, moving the supernate and the DNA binding solution to a sleeve tube with a silicon dioxide membrane fixed thereon, performing centrifugation with a liquid being abandoned, and eluting the DNA. The method is simple and easy to carry out, is free of toxic and side effects and is easy to be popularized.
Description
Technical field
The present invention relates to the method for DNA in a kind of rapid extraction and purifying formalin-fixed tissue, belong to medical jurisprudence technical field.
Background technology
Formalin-fixed tissue is pathology, the subject such as forensic pathology and medicolegal genetics carries out medical diagnosis on disease and sample is commonly used in case analysis, these samples all need fix several months and even several years in formalin, as a kind of important individual archival objects, be accused of the civil or criminal case such as medical tangle, Insurance Fraud, the inheritance of property provide a kind of very important, forensic sample reliably for solving.When but general extraction methods carries out gene type to formalin-fixed tissue, be difficult to obtain stable, reliable genotyping result, this is because formalin has an impact to specimen dna quality and amplification, cause the specimen dna Quality Down extracted, and then make the generation of pcr amplification product and follow-up biological analysis become difficulty.
Formalin has 4 kinds of chemically modified effects to DNA molecular: 1) nucleic acid methylation effect, 2) formed due to methylation crosslinked, 3) induced synthesis purine dimer, 4) cause phosphodiester bond fracture in nucleic acid.Formalin causes DNA generation degraded in various degree by the nucleic acid molecule arrangement changing sample tissue.Crosslinked between DNA and protein, between protein-protein, between DNA and DNA that the factor that DNA quality is extracted in impact from formalin-fixed sample comprises that formalin causes, the chemical composition of formalin solution, pH value and concentration, the time of Saving specimen and temperature, Saving specimen position etc.In addition, the DNA degradation of formalin fixed preparation become the fragment of different lengths to be also unfavorable for specimen dna carries out pcr amplification reaction.Such as small pieces segment DNA and large fragment DNA compete Taq polysaccharase jointly, and small pieces segment DNA cannot effectively increase, and therefore PCR reaction is suppressed.Formalin also can cause N-glycosyl hydrolase in DNA, produces non-purine and non-pyrimidine site, and a large amount of non-purine and non-pyrimidine site are gathered in guiding region and cause primer cannot normally be connected with DNA profiling.
Blowing gently auspicious etc., it is a kind of from soaking the method (" extracting method of the zoological specimens genomic dna of formalin-fixed " extracting genomic dna the Cricetulus trition liver of formalin and Japanese eel muscle specimen to propose, animal journal, 2002,48 (2): 264 ~ 269).Get and be soaked in Cricetulus trition liver in formalin or Japanese eel muscle is appropriate, rinse by PBS solution, the thieving paper being placed on sterilizing is wiped dry, in Bechtop, by sterile scissors, material is cut into the fritter of 50mg, puts into PBS solution and rinse; Then proceed in the ethanol of 70% and process 12 ~ 24h.Change to successively in following graded ethanol and process: 80% ethanol, 2h, repeat once; 90% ethanol, 2h, repeats once; 95% ethanol, 2h, repeats once; 100% ethanol, 1h, repeats once.Then PBS liquid material being put into 1/2 times soaks 12h, and period changes a solution.Extracting method is with reference to people (1989) such as Sambroock, add the amount of Proteinase K according to standard amount (100 μ g/mL), in 50 ~ 56 DEG C of temperature bath 3 ~ 6h treating processess, continuous jog mixing, 1/2 times of Proteinase K that can repeat to add normal content depending on digestion effect digests, till by material complete digestion; The last supernatant liquor successively of phenol chloroform moves in dialysis tubing dialyses; During precipitation DNA, at-20 DEG C, 20min effect is advisable.The principal feature of the method is to carry out pre-treatment to sample, ensureing under the prerequisite not making DNA degrade further, first remove formalin composition contained in sample, then utilize the phenol chloroform extraction method of improvement to extract the genomic dna of such sample, then use dialysis method purify DNA further.Result of study shows, adopts the method Isolation and purification tested sample genomic dna can be applied to RAPD, the pcr amplification of microsatellite locus, Southern and dot hybridization preferably.But the method for there is no meets the genomic dna of medical jurisprudence laboratory needs.
Summary of the invention
The object of this invention is to provide the method for DNA in a kind of rapid extraction and purifying formalin-fixed tissue.
In order to realize above object, the technical solution adopted in the present invention is:
A method of DNA in rapid extraction and purifying formalin-fixed tissue, comprises the following steps:
(1) get formalin-fixed tissue, disrupting tissue after distilled water flushing, blots with filter paper;
(2) in the tissue of step (1), soaked in absolute ethyl alcohol is added 1 ~ 5 minute, centrifugal, supernatant discarded; Add the ethanol of concentration 70 ~ 80% and the ethanol of concentration 40 ~ 60% more successively, repeat above step, finally leave and take precipitation;
(3) add cell pyrolysis liquid, sodium laurylsulfonate and Proteinase K in the precipitation left and taken in step (2), at temperature 55 ~ 65 DEG C, hatching process, centrifugal, gets supernatant liquor;
(4) in the supernatant liquor of step (3), salt is added, mixing, stratification at temperature-4 ~ 4 DEG C, centrifugal, get supernatant liquor;
(5) in the supernatant liquor of step (4), add DNA in conjunction with liquid, mixing, transfers to and is fixed with in the sleeve pipe of silicon dioxide film, centrifugal, discards liquid, eluted dna, to obtain final product.
Slightly can shake when soaking in described step (2), ensure that ethanol fully contacts broken tissue.
Rotating speed centrifugal in described step (2) is 10000 ~ 12000rpm, centrifugation time 3 ~ 5 minutes.
Naturally hang 3 ~ 5 minutes after leaving and taking precipitation in described step (2), give full play to make ethanol.
In described step (3), cell pyrolysis liquid consists of: NaCl 0.4mol/L, Tris-HCl 0.01mol/L, EDTA 0.002mol/L, surplus is water.
In described step (3) when sodium laurylsulfonate concentration get 10%, Proteinase K get 20mg/mL time, the volume ratio of cell pyrolysis liquid and sodium laurylsulfonate, Proteinase K is 40:2:1.As when the quality of fixing organization is 20 ~ 30mg, cell pyrolysis liquid gets 400 μ L, and 10% sodium laurylsulfonate gets 20 μ L, and 20mg/mL Proteinase K gets 10 μ L.
In described step (3), the time of hatching process is 8 ~ 10 hours, until solution is limpid.
At room temperature place (as 15 ~ 30 minutes) after hatching process in described step (3), treat that nature is down to room temperature.
Rotating speed centrifugal in described step (3) is 11000 ~ 15000rpm, centrifugation time 8 ~ 15 minutes.
In described step (4), salt is sodium-chlor, as the sodium chloride solution of high density, and 5mol/L NaCl.
The time left standstill in described step (4) is 5 ~ 15 minutes, layering to appear.
The temperature left standstill in described step (4) is preferably 0 DEG C.
In described step (5), DNA consists of in conjunction with liquid: 5mol/L Guanidinium hydrochloride, 35% ethanol.
In described step (5), silicon dioxide film and sleeve pipe are commercial goods (silicon-dioxide film preparation test kit), purchased from the clean biochemical technology company limited of Hangzhou Wei Te.
Rotating speed centrifugal in described step (5) is 3000 ~ 4000rpm, centrifugation time 1 ~ 5 minute.
In described step (5) before eluted dna, concentration 70 ~ 80% washing with alcohol sleeve pipe need be adopted, then use distilled water eluted dna.As added 75% ethanol, discard liquid (repetitive operation 1 time) after vortex oscillation, then add distilled water, centrifugal eluted dna, centrifugal rotating speed is 12000 ~ 18000rpm, centrifugation time 1 ~ 5 minute.
Be placed in-20 ~ 4 DEG C through the DNA of wash-out in described step (5) to save backup.
Beneficial effect of the present invention:
In Isolation and purification formalin-fixed tissue of the present invention, method is simple, have no side effect for DNA, is easy to promote, and meets the demand in medical jurisprudence laboratory.Adopt the method to obtain DNA and can be used for STR composite amplification.Meanwhile, the present invention is in the operation of removing formalin composition contained by fixing organization, and adopt concentration ethanol from high to low to process, the removal effect of PARA FORMALDEHYDE PRILLS(91,95) is better.
Accompanying drawing explanation
Fig. 1 is that in test example of the present invention, formalin-fixed tissue extraction DNA divides capillary electrophoresis figure through STR composite amplification;
Fig. 2 is that in test example, same individuality refers to that blood DNA divides capillary electrophoresis figure through STR composite amplification with fixing organization;
Fig. 3 is that test example positives contrast STR composite amplification divides capillary electrophoresis figure (9947A);
Fig. 4 is that in test example, negative control STR composite amplification divides capillary electrophoresis figure;
Fig. 5 is test example allelic somatotype standard substance (ID Ladder).
Embodiment
Following embodiment is only described in further detail the present invention, but does not form any limitation of the invention.
Embodiment 1
The method of DNA in rapid extraction and purifying formalin-fixed tissue in the present embodiment, comprises the following steps:
(1) get formalin fixing human cadaveric tissue 25mg (as peanut size 20 ~ 30mg), put in 5mL test tube, add ddH
2o 3mL, washes 3 times repeatedly, and take out tissue, shredded by tissue (shredding), filter paper blots for subsequent use as far as possible;
(2) in the tissue of step (1), add dehydrated alcohol (100%) soak, slight oscillatory 1 minute, ensure that ethanol fully contacts disrupting tissue, centrifugal 3 minutes of 12000rpm, stays precipitation to remove supernatant; Add the ethanol of concentration 75% and the ethanol of 50% successively, repeat above step, finally leave and take precipitation, naturally hang 3 minutes for subsequent use;
(3) in the precipitation of step (2), add cell pyrolysis liquid 400 μ L, 10%SDS 20 μ L and 20mg/ml PK 10 μ L, incubated overnight at temperature 56 DEG C, until solution is limpid, take out and at room temperature place 20 minutes, under rotating speed 13000rpm centrifugal 10 minutes, get supernatant liquor for subsequent use;
The formula of cell pyrolysis liquid is:
0.4mol/L NaCL(58.44) 11.7g(11.688g)，
1 mol/L TrisHcl 5ml，
0.1 mol/L EDTA 10ml，
Add distilled water to 500ml DW, sterilization, 4 DEG C of preservations;
(4) in the supernatant liquor of step (3), add 5mol/L NaCl 200 μ L, mixing, at temperature 0 DEG C, leave standstill 10 minutes, occur layering, centrifugal, get supernatant liquor;
(5) in the supernatant liquor of step (4), DNA is added in conjunction with liquid 450 μ L (5mol/L Guanidinium hydrochloride, 35% ethanol), mixing, transfer to the sleeve pipe interior (purchased from the clean biochemical technology company limited of Hangzhou Wei Te) being fixed with silicon dioxide film, under rotating speed 3600rpm centrifugal 1 minute, inhale and abandon liquid; In sleeve pipe, add the ethanol 500 μ L of concentration 75%, after vortex oscillation, inhale and abandon liquid, repetitive operation 1 time; DdH is added in sleeve pipe
2o 50 μ L, under rotating speed 15000rpm centrifugal 1 minute, eluted dna, 4 DEG C saved backup.
Embodiment 2
The method of DNA in rapid extraction and purifying formalin-fixed tissue in the present embodiment, comprises the following steps:
(1) get formalin fixing human organ-tissue 20mg, put in 5mL test tube, add ddH
2o 3mL, washes 3 times repeatedly, and take out tissue, shredded by tissue (shredding), filter paper blots for subsequent use as far as possible;
(2) in the tissue of step (1), add dehydrated alcohol (100%) soak, slight oscillatory 1 minute, ensure that ethanol fully contacts disrupting tissue, centrifugal 5 minutes of 10000rpm, stays precipitation to remove supernatant; Add the ethanol of concentration 70% and the ethanol of 40% successively, repeat above step, finally leave and take precipitation, naturally hang 5 minutes for subsequent use;
(3) in the precipitation of step (2), add cell pyrolysis liquid 400 μ L (same embodiment 1 of filling a prescription), 10%SDS 20 μ L and 20mg/ml PK 10 μ L, incubated overnight under temperature 60 C, until solution is limpid, take out and at room temperature place 15 minutes, under rotating speed 11000rpm centrifugal 15 minutes, get supernatant liquor for subsequent use;
(4) in the supernatant liquor of step (3), add 5mol/L NaCl 200 μ L, mixing, at temperature-4 DEG C, leave standstill 5 minutes, occur layering, centrifugal, get supernatant liquor;
(5) in the supernatant liquor of step (4), DNA is added in conjunction with liquid 450 μ L (5mol/L Guanidinium hydrochloride, 35% ethanol), mixing, transfer to the sleeve pipe interior (purchased from the clean biochemical technology company limited of Hangzhou Wei Te) being fixed with silicon dioxide film, under rotating speed 3000rpm centrifugal 2 minutes, inhale and abandon liquid; In sleeve pipe, add the ethanol 500 μ L of concentration 75%, after vortex oscillation, inhale and abandon liquid, repetitive operation 1 time; DdH is added in sleeve pipe
2o 50 μ L, under rotating speed 12000rpm centrifugal 5 minutes, eluted dna, 4 DEG C saved backup.
Embodiment 3
The method of DNA in rapid extraction and purifying formalin-fixed tissue in the present embodiment, comprises the following steps:
(1) get formalin fixing human cadaveric tissue 30mg, put in 5mL test tube, add ddH
2o 3mL, washes 3 times repeatedly, and take out tissue, shredded by tissue (shredding), filter paper blots for subsequent use as far as possible;
(2) in the tissue of step (1), add dehydrated alcohol (100%) soak, slight oscillatory 1 minute, ensure that ethanol fully contacts disrupting tissue, centrifugal 5 minutes of 10000rpm, stays precipitation to remove supernatant; Add the ethanol of concentration 80% and the ethanol of 60% successively, repeat above step, finally leave and take precipitation, naturally hang 4 minutes for subsequent use;
(3) in the precipitation of step (2), add cell pyrolysis liquid 400 μ L (same embodiment 1 of filling a prescription), 10%SDS 20 μ L and 20mg/ml PK 10 μ L, incubated overnight at temperature 65 DEG C, until solution is limpid, take out and at room temperature place 30 minutes, under rotating speed 15000rpm centrifugal 8 minutes, get supernatant liquor for subsequent use;
(4) in the supernatant liquor of step (3), add 5mol/L NaCl 200 μ L, mixing, at temperature 4 DEG C, leave standstill 15 minutes, occur layering, centrifugal, get supernatant liquor;
(5) in the supernatant liquor of step (4), DNA is added in conjunction with liquid 450 μ L (5mol/L Guanidinium hydrochloride, 35% ethanol), mixing, transfer to the sleeve pipe interior (purchased from the clean biochemical technology company limited of Hangzhou Wei Te) being fixed with silicon dioxide film, under rotating speed 4000rpm centrifugal 1 minute, inhale and abandon liquid; In sleeve pipe, add the ethanol 500 μ L of concentration 75%, after vortex oscillation, inhale and abandon liquid, repetitive operation 1 time; DdH is added in sleeve pipe
2o 50 μ L, under rotating speed 18000rpm centrifugal 1 minute, eluted dna ,-20 DEG C saved backup.
Test example
The DNA of Example 1 Isolation and purification, with fixing organization with integrally referring to blood DNA, positive control (Human Cell Line DNA 9947A, Applied
life Technologies Corporation), negative control (ddH
2and allelic ladder (ID Ladder, Applied O)
life Technologies Corporation), after STR composite amplification, carry out capillary electrophoresis (with reference to Applied
the Protocols of Life Technologies Corporation), test-results refers to Fig. 1 ~ Fig. 5.(test method is described in suggestion in detail, as the method for DNA through STR composite amplification, the method for capillary electrophoresis)
Result shows: allelic ladder, positive control and negative control are all correct, and the DNA that fixing organization extracts is through Identifiler kit (Applied
life Technologies Corporation) STR composite amplification reagent kit is analysing amplified, assert fixing organization and refers to that blood derives from same individuality, illustrates that DNA that the method is extracted can meet the demand in medical jurisprudence laboratory.
Claims (10)
1. a method of DNA in rapid extraction and purifying formalin-fixed tissue, is characterized in that: comprise the following steps:
(1) get formalin-fixed tissue, disrupting tissue after distilled water flushing, blots with filter paper;
(2) in the tissue of step (1), soaked in absolute ethyl alcohol is added 1 ~ 5 minute, centrifugal, supernatant discarded; Add the ethanol of concentration 70 ~ 80% and the ethanol of concentration 40 ~ 60% more successively, repeat above step, finally leave and take precipitation;
(3) add cell pyrolysis liquid, sodium laurylsulfonate and Proteinase K in the precipitation left and taken in step (2), at temperature 55 ~ 65 DEG C, hatching process, centrifugal, gets supernatant liquor;
(4) in the supernatant liquor of step (3), salt is added, mixing, stratification at temperature-4 ~ 4 DEG C, centrifugal, get supernatant liquor;
(5) in the supernatant liquor of step (4), add DNA in conjunction with liquid, mixing, transfers to and is fixed with in the sleeve pipe of silicon dioxide film, centrifugal, discards liquid, eluted dna, to obtain final product.
2. the method for DNA in rapid extraction according to claim 1 and purifying formalin-fixed tissue, is characterized in that: rotating speed centrifugal in described step (2) is 10000 ~ 12000rpm, centrifugation time 3 ~ 5 minutes.
3. the method for DNA in rapid extraction according to claim 1 and purifying formalin-fixed tissue, it is characterized in that: in described step (3), cell pyrolysis liquid consists of: NaCl 0.4mol/L, Tris-HCl 0.01mol/L, EDTA 0.002mol/L, surplus is water.
4. the method for DNA in rapid extraction according to claim 1 and purifying formalin-fixed tissue, is characterized in that: in described step (3), the time of hatching process is 8 ~ 10 hours.
5. the method for DNA in rapid extraction according to claim 1 and purifying formalin-fixed tissue, is characterized in that: rotating speed centrifugal in described step (3) is 11000 ~ 15000rpm, centrifugation time 8 ~ 15 minutes.
6. the method for DNA in rapid extraction according to claim 1 and purifying formalin-fixed tissue, is characterized in that: in described step (4), salt is sodium-chlor.
7. the method for DNA in rapid extraction according to claim 1 and purifying formalin-fixed tissue, is characterized in that: the time left standstill in described step (4) is 5 ~ 15 minutes.
8. the method for DNA in rapid extraction according to claim 1 and purifying formalin-fixed tissue, is characterized in that: in described step (5), DNA consists of in conjunction with liquid: 5mol/L Guanidinium hydrochloride, 35% ethanol.
9. the method for DNA in rapid extraction according to claim 1 and purifying formalin-fixed tissue, is characterized in that: rotating speed centrifugal in described step (5) is 3000 ~ 4000rpm, centrifugation time 1 ~ 5 minute.
10. the method for DNA in rapid extraction according to claim 1 and purifying formalin-fixed tissue, it is characterized in that: in described step (5) before eluted dna, concentration 70 ~ 80% washing with alcohol sleeve pipe need be adopted, then use distilled water eluted dna.
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Cited By (2)
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| CN105424429A (en) * | 2015-11-04 | 2016-03-23 | 哈尔滨医科大学 | Sample pretreatment method for formalin fixation tissue non-target metabonomics research |
| CN107922940A (en) * | 2015-03-12 | 2018-04-17 | 财团法人峩山社会福祉财团 | The method of seperated nuclear acid from FFPE tissues |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107922940A (en) * | 2015-03-12 | 2018-04-17 | 财团法人峩山社会福祉财团 | The method of seperated nuclear acid from FFPE tissues |
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