CN101613696B - Reagent for extracting and purifying DNA - Google Patents
Reagent for extracting and purifying DNA Download PDFInfo
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- CN101613696B CN101613696B CN2009100903356A CN200910090335A CN101613696B CN 101613696 B CN101613696 B CN 101613696B CN 2009100903356 A CN2009100903356 A CN 2009100903356A CN 200910090335 A CN200910090335 A CN 200910090335A CN 101613696 B CN101613696 B CN 101613696B
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Abstract
The invention discloses a reagent for extracting and purifying DNA, comprising the following substances: preprocessing lysis solution with pH of 7.4-8.5: water serves as solvent, and solute comprises the substances with the following final concentration: 10-100mM of buffer salt, 0.5%-5% of surface active agent, 1-100mM of chelating agent, 50-150mM of soluble salt, and 0.2-4mg/ml of protease K; pyrolysis combined liquid with pH of 6.4-7.4: water serves as solvent, and solute comprises the substances with the following final concentration: 20-150mM of buffer salt, 3-8 of MChaotropic salt, 1-10%of surface active agent, 0.5-4% of amphoteric ion detergent, 10-100mM of chelating agent and 15-30% of alcohol; cleaning solution I: water serves as solvent, and solute comprises the substances with the following final concentration: 4-6 of MChaotropic salt, 25-50% of alcohol and 10-100mM of chelating agent; cleaning solution II is 75% of ethanol water solution; spent regenerant is TE (10mM Tris-HCl, 1mMEDTA, and pH is 8.0); magnetic bead suspension is nanoscale silicone coated magnetic microsphere (50-100mg/ml).The DNA extracting efficiency is as high as 93.67% by adopting the reagent.
Description
Technical field
The present invention relates to biological technical field, particularly a kind of reagent that extracts purify DNA.
Background technology
The DNA separation and purification is very important operation in the molecular biology experiment, and can the quality of nucleic acid be directly connected to subsequent operations and carry out smoothly.The development of biology, medical jurisprudence and genomics has been strengthened obtain the demand of the complicated approach of nucleic acid from various biological materials.For example, thymus nucleic acid provides widely about genetic origin and genetic polymorphism information.These information can be used for medicolegal genetics and identify practice.
Existing various nucleic acid purification methods all can be divided into following two big classes at present: liquid phase purifying and solid phase purifying.In the liquid phase purifying, nucleic acid is maintained liquid state, and impurity is by precipitation, centrifugal be removed or nucleic acid is precipitated comes out, and impurity is retained; In the solid phase purifying, nucleic acid is incorporated on the solid phase carrier, and impurity is by the selectivity wash-out.For example, adopt the liquid phase purifying to come separated DNA to be retained in the liquid phase, and impurity by by such as precipitation and/or centrifugal method remove.DNA is attached on the solid phase carrier in the solid phase purifying, and impurity such as RNA, protein and phosphatide etc. are by the selectivity wash-out.In the DNA purifying, if parent material comprises cell, liquid phase process and solid phase method all need cell or virus to break altogether, or cleavage step.Break or cleavage step produces and blended DNA such as pollutent such as RNA, lipid, carbohydrate, protein.This mixture also contains must being removed of degradation of dna and/or deactivation not infect the generation DNA enzyme of undegradable DNA basically.Two big classes of purifying were all utilized conventional method in the past, needed trivial step, and used deleterious reagent usually, had also occurred extracting method faster now, as the Chelex-100 method, only needed less step, and common less harmful reagent.
A kind of quick liquid phase DNA purification process of Chelex-100 method, Chelex-100 is a kind of chemical finishing resin, is made up of vinylbenzene, divinylbenzene interpolymer.Contain paired Iminodiacetate ion, integrate polyvalent metal ion, especially selectivity is integrated divalent ion, has higher Metal Ion Selective Electrode and stronger bonding force than conventional ion exchanger. and can be in conjunction with many other allogenic materials that may influence step analysis.By the centrifugal Chelex-100 particle of removing, these are separated with DNA with Chelex-100 bonded material, and the inhibitor or the impurity that prevent to be attached among the Chelex take in the PCR reaction, influence next step DNA analysis, and, prevent dna degradation by bind metal ion.Utilize sequestrant to remove metallic impurity in the biological sample and reach the purpose of purifying.This method is at first used the deionized water wash biological sample, and the suspension that adds resin is hatched certain hour for 56 ℃, hatches 8 minutes for 100 ℃, then by centrifugal removal impurity.This method simple, quick (only relating to the use of two kinds of reagent).But it is not enough that this method is extracted the DNA purity that obtains, and can not satisfy the requirement of minim DNA biological sample DNA extraction purifying.
Solid phase extractions method development in recent years is rapid, occupies important status in the nucleic acid extraction field.These class methods generally need four key steps: lysing cell breaks with released dna cytolemma, nuclear membrane DNA; The DNA that discharges combines with solid phase carrier; The washing of impurity; Wash-out and obtain the DNA of purifying.For solid phase DNA purifying, used multiple solid phase carrier, as silicon dioxide microsphere, membrane filter, metal oxide, latex precipitation, diatomite, glass powder etc.When nucleic acid is present in high density chaotropic salt and low pH solution, can be adsorbed onto silicon class dielectric surface, and in the less salt high pH solution, elute, extract the purifying purpose thereby reach.Vogelstein directly uses glass powder to extract DNA from sepharose, and Marko uses glass powder success purifying DNA plasmid.Boom etc. are improved this method, are solid-phase adsorbent with silicon-dioxide and diatomite, are used for extracting the micro-pathogen gene group DNA in purifying human serum or the urine.This method needs centrifugal, five kinds of reagent purify DNA from whole blood of six steps.
Summary of the invention
The object of the present invention is to provide a kind of reagent that extracts purify DNA.
The reagent of extraction purify DNA provided by the invention, form by following material:
Pre-treated lysis solution: pH is 7.4-8.5, and solvent is a water, and solute is the material of following final concentration: 10-100mM buffering salt, the tensio-active agent of 0.5-5g/100ml, the sequestrant of 1-100mM, the soluble salt of 50-150mM and the Proteinase K of 0.2-4mg/ml;
Cracking is in conjunction with liquid: pH=6.4-7.4, solvent is a water, solute is the buffering salt of the material of following final concentration: 20-150mM, the Chaotropic salt of 3-8M, the tensio-active agent of 1-10% (volumn concentration), 0.5-4g/100ml zwitterionic detergent, the alcohol of the sequestrant of 10-100mM and 15-30% (volumn concentration);
Washings I: solvent is a water, and solute is the Chaotropic salt of the material of following final concentration: 4-6M, the sequestrant of the pure and mild 10-100mM of 25-50% (volumn concentration);
Cleaning solution II: 75-80% (volumn concentration) aqueous ethanolic solution;
Elutriant: be TE solution, the pH of described TE solution is 8.0, and solvent is a water, and solute is the Tris-HCl of 0-20mM, the EDTA of 0-2mM;
Bead suspension: the composition of every ml bead suspension is as follows: diameter be the nano silicone bag of 50-1200nm by magnetic microsphere 50-100mg, all the other are water.
The extraction of DNA in seminal stain and the hair needs to add DTT (dithiothreitol (DTT)) in pre-treated lysis solution, and in the above-mentioned pre-treated lysis solution, solute also comprises the dithiothreitol (DTT) of 0.025-0.05M.So the another kind of reagent that extracts purify DNA provided by the invention is made up of following material:
Pre-treated lysis solution: pH is 7.4-8.5, solvent is a water, solute is the material of following final concentration: the 10-100mM buffering salt, 0.5-5g/100ml tensio-active agent, the sequestrant of 1-100mM, the dithiothreitol (DTT) of the soluble salt of 50-150mM and the Proteinase K of 0.2-4mg/ml and 0.025-0.05M;
Cracking is in conjunction with liquid: pH=6.4-7.4, solvent is a water, solute is the buffering salt of the material of following final concentration: 20-150mM, the Chaotropic salt of 3-8M, the tensio-active agent of 1-10% (volume percent), 0.5-4g/100ml zwitterionic detergent, the alcohol of the sequestrant of 10-100mM and 15-30% (volumn concentration);
Washings I: solvent is a water, and solute is the Chaotropic salt of the material of following final concentration: 4-6M, the sequestrant of the pure and mild 10-100mM of 25-50% (volumn concentration);
Cleaning solution II: 75-80% (volumn concentration) aqueous ethanolic solution;
Elutriant: be TE solution, the pH of described TE solution is 8.0, and solvent is a water, and solute is the Tris-HCl of 0-20mM, the EDTA of 0-2mM;
Bead suspension: the composition of every ml bead suspension is as follows: diameter be the nano silicone bag of 50-1200nm by magnetic microsphere 50-100mg, all the other are water.
The concentration of above-mentioned dithiothreitol (DTT) is 0.04M-0.05M preferably.
In above-mentioned all pre-treated lysis solution, the concentration of buffering salt is preferably 20-80mM, and surfactant concentrations is preferably 0.5-4g/100ml, and the concentration of sequestrant is preferably 20-80mM, the concentration of soluble salt is preferably 50-130mM, and the concentration of Proteinase K is preferably 0.2-2mg/ml; Described soluble salt is 80-100mM more preferably, and Proteinase K is 0.2-1mg/ml more preferably; Tensio-active agent in the described pre-treated lysis solution is anion surfactant or nonionogenic tenside, anion surfactant preferably, and described anion surfactant is sodium laurylsulfonate or sarcosyl; Described Digestive system also comprises a kind of soluble salt solution, and DNA discharges from nucleus and with after histone separates, needs to improve the solubleness of DNA in solution.The solubleness of DNA in the finite concentration sodium chloride solution is very high, and Na+ and electronegative DNA are combined into the DNA sodium salt.This makes DNA be dissolved state in solution and keeps the stability of DNA in solution.In certain embodiments, can use soluble salts such as NaCl.
Proteinase K is the wider serine protease of a kind of nicking activity.The carboxy-terminal peptide bond of its cutting aliphatic amino acid and die aromatischen Aminosaeuren is used for the proteinic general degraded of biological sample.Purified RNA enzyme and the DNA enzymic activity removed of this enzyme.Because Proteinase K is stable in urea and SDS, also has the ability of degraded natural protein, thereby it uses very extensive
Various biological materials at first will soak through pre-treated lysis solution as blood cake, seminal stain, stub, hair, tissue etc., and after hatching 0.5h-4h under 50-65 ℃ the temperature, can carry out the subsequent extracted operation.The temperature of hatching is preferably 56-60 ℃.
By the pre-treatment of pre-treated lysis solution to biological specimen, the one, adherent cell on the carrier (as blood leukocytes, spermatid, epithelial cell etc.) is separated with carrier with DNA or with tissue in break away from, be free in the pre-treated lysis solution; The 2nd, lysing cell is released in the Digestive system DNA and pollutents such as protein, phosphatide, promptly forms thick lysate and finishes the sample pre-treatment after removing carrier by method such as centrifugal.
Comprise cell contamination things such as DNA and protein, cell debris, phosphatide in the thick lysate that forms after the pre-treatment.For lysing cell more fully, DNA is discharged from cell, and the DNA that discharges is combined with magnetic microsphere to reach the purpose of separation and purification, can use cracking in conjunction with liquid.
Above-mentioned cracking is in conjunction with in the liquid, the concentration of Chaotropic salt is preferably 4-6M, the concentration of buffering salt is preferably 50-120mM, the concentration of sequestrant is preferably 20-80mM, surfactant concentrations is preferably 2-8% (volume percent), the concentration of zwitterionic detergent is preferably 1-3g/100ml, and the concentration of alcohol is preferably 20-28% (volumn concentration); Described cracking is preferably used nonionogenic tenside in conjunction with the tensio-active agent in the liquid, and described nonionogenic tenside is Triton X-100, Tween 20, Tween 21, Tween.40, Tween 60, Tween 80, Tween 85, NP-40, Brij 30 or Span 20; Described zwitterionic detergent is 3-(3-cholamine propyl group)-dimethylamino-1-propanesulfonic acid; Described alcohol is ethanol, Virahol or polyoxyethylene glycol.
Among the above-mentioned washings I, the concentration of Chaotropic salt is preferably 4.2-5.8M, and alcohol is the ethanol of 30-45% (volumn concentration), and the concentration of sequestrant is preferably 20-80mM.
Above-mentioned cleaning solution II is preferably: 75% aqueous ethanolic solution.
Above-mentioned washings I and II are used for washing the complex body that the DNA-magnetic Nano microsphere forms, to remove and pollutent such as the protein, phosphatide of magnetic bead bonded except that nucleic acid.
The preferred 1mM of concentration of the preferred 10mM of the concentration of Tris-HCl in the described elutriant, EDTA, described elutriant pH is preferably 8.0.Remove with pollutents such as solid phase carrier bonded protein, phosphatide after, must be eluted to solid phase carrier bonded DNA and just can be applied to follow-up forensic dna analysis in the aqueous phase solution.DNA-magnetic microsphere complex body can make DNA separate with solid phase carrier with after elutriant contacts.
Elutriant can be the aqueous phase solution of low ionic strength, also can be the aqueous solution of deionized water or the low ionic strength of certain PH.The elutriant pH value must can guarantee dna stability, integrity.Can be lower concentration TE (10mM Tris-HCl, 1mM EDTA, pH8.0) wash-out and solid phase carrier bonded DNA.
Nano silicone bag in above-mentioned every ml bead suspension is preferably 50mg by magnetic microsphere.
In mentioned reagent, described buffering salt is Tris-HCl, phosphoric acid salt or HEPES salt.
Above-mentioned Chaotropic salt has high-dissolvability in aqueous solution.The Chaotropic salt of high density can cause that protein opens folding, as to destroy nucleic acid secondary structure and make the protein and the double-stranded DNA dissolving of sex change.It has been generally acknowledged that, this is because the Chaotropic salt ion can destroy the hydrogen bond network in its place solution system, higher thermodynamic stability when making the protein of sex change and denatured DNA show than its correct folding or native conformation, and can guarantee to allow DNA preferentially be attached on the magnetic Nano microsphere.This Chaotropic salt can be any known Chaotropic salt, as guanidinium isothiocyanate, guanidine thiocyanate, Guanidinium hydrochloride, urea, sodium iodide, potassiumiodide, lithium chloride, sodium perchlorate, Trichloroacetate.What use in the invention is guanidinesalt (as guanidinium isothiocyanate, guanidine thiocyanate, Guanidinium hydrochloride).Especially guanidinium isothiocyanate, better lysing cell and suppress the activity of nuclease is a unusual effective dna purified reagent in.Chaotropic salt can 3～8M concentration be present in cracking in conjunction with in the liquid.In any reagent that contains Chaotropic salt of the present invention, the concentration of Chaotropic salt must be lower than the solubleness in this reagent of its place.The concentration of certain C haotropic salt can influence DNA and combine with magnetic Nano microsphere.Chaotropic salt must reach certain concentration just can impel combining of DNA and magnetic microsphere.But too high salt concn can make protein denaturation and dna degradation, and is precipitated out from solution.Protein and macromolecular double-stranded DNA when the Chaotropic salt concn is 0.5～2M stable existence in solution.On the contrary, RNA, small molecule DNA such as single stranded DNA, dna fragmentation are stablized pure being in the solution system when concentration is 2～5M.
Above-mentioned sequestrant be a kind of can with Na
+, Mg
2+, Ca
2+, Mn
2+, Zn
2+The mixture that combines Deng metal ion.Have at least two nonmetallic ion to combine in the sequestrant, will no longer be free in the solution system with sequestrant bonded metal ion with free metal positively charged ion formation covalent linkage and with it.When having sequestrant to exist in the solution system, can reduce GOLD FROM PLATING SOLUTION and belong to ionic concentration.The reduction of free metal ion concentration makes the nuclease inactivation, can effectively prevent DNA by nuclease degradation, play the effect of protection DNA integrity.A lot of operational sequestrants are not limited only to EDTA, EGTA, CDTA.
The test kit that above-mentioned reagent is made also belongs within protection scope of the present invention.
Reagent provided by the present invention and test kit can be realized extracting from all kinds biological specimen (contact sample, corrupt outmoded samples such as blood, blood cake, seminal fluid, seminal stain, hair, tissue, vaginal swab, buccal swab, salivary stain, sweat spot, cast-off cells), purify DNA.And can satisfy the extraction requirement of the sample of routine and minim DNA simultaneously.Utilize the DNA of reagent purifying gained of the present invention to need not to be further purified, promptly can be applicable to downstream analysis operations such as STR composite amplification, dna sequencing, DNA be quantitative.The DNA that reagent of the present invention and cartridge system thereof are extracted is particularly suitable for being applied to pcr amplification (STR, dna sequencing, real-time fluorescence quantitative PCR etc.).
Beneficial effect of the present invention is as follows:
Integrated operation is simple, quick;
The biological sample scope of application widely can realize the DNA extraction of most of biological samples;
Lytic effect is good, and the abundant cracking that guarantees cell is with released dna;
The purifying ability is strong, can obtain enough pure in the sample that is mixed with a large amount of PCR inhibitor or pollutent and can be satisfied with the DNA of operation such as downstream PCR, dna sequencing, real time fluorescent quantitative fully;
The extraction efficiency height.Can reach the extraction efficiency more than 90%, maximization avoids DNA in the loss of extracting operating process; The extraction of the DNA standard substance that reagent that this invention is provided and method are applied to concentration known, through whole leaching process, (the DNA amount (ng) that wash-out obtains/adding DNA standard substance (G1471, Promega company) total amount (ng) can effectively improve the STR somatotype success ratio of minim DNA biological samples such as trace, degraded to calculate extraction efficiency by following formula;
Reagent system by enlarging the operation system of reagent, can be realized the enrichment of contained minim DNA sample in the large volume sample flexibly;
Entire operation can be avoided centrifugally operated, applicablely is used for various DNA automatizations and extracts service platforms.
Description of drawings
The compliance test result figure that Fig. 1 extracts DNA for reagent and the method for employing embodiment 1, wherein Figure 1A is the DNA STR somatotype collection of illustrative plates of micro blood, Figure 1B is the quantitative fluorescent PCR electrophoretogram.
Fig. 2 is 3 * 3mm blood cake (FTA card) STR somatotype collection of illustrative plates.
Fig. 3 is blood cake on the cotton swab (2ul anticoagulation) STR somatotype collection of illustrative plates.
Fig. 4 is that liquid anticoagulation of 2 μ l and PCR inhibitor etc. are blended in the STR collection of illustrative plates that forms blood cake on the colourless cotton.
Fig. 5 is that the liquid anticoagulation of 2 μ l drops in the STR somatotype collection of illustrative plates that jeans form blood cake.
Fig. 6 is 1 μ l seminal fluid forms seminal stain on colourless cotton a STR somatotype collection of illustrative plates.
Fig. 7 is the STR somatotype collection of illustrative plates that 50 μ l salivas drop in the buccal swab that forms on the cotton swab.
Fig. 8 is a stub STR somatotype collection of illustrative plates.
Fig. 9 is the hair STR somatotype collection of illustrative plates that comes off naturally.
Figure 10 is the DNATyper of tissue piece
TM15STR somatotype collection of illustrative plates.
Figure 11 is the cast-off cells STR somatotype collection of illustrative plates on the mobile phone.
Figure 12 is the cast-off cells STR somatotype collection of illustrative plates on the sweater.
Figure 13 is a cast-off cells STR somatotype collection of illustrative plates on the wallet.
Figure 14 is a rim of a cup cast-off cells STR somatotype collection of illustrative plates.
Figure 15 is a keyboard cast-off cells STR somatotype collection of illustrative plates.
Figure 16 is corrupt blood cake STR somatotype collection of illustrative plates.
Figure 17 is the STR of a corrupt tissue somatotype collection of illustrative plates.
Embodiment
The invention will be further described below in conjunction with specific embodiment, but the present invention is not limited to following examples.
Among the following embodiment, if no special instructions, be ordinary method.
DNA and compliance test result in embodiment 1, the extraction purifying micro blood
One, DNA and the STR composite amplification that extracts in the purifying micro blood analyzed
1, reagent
Reagent is as follows:
Pre-treated lysis solution: pH is 7.4, and solvent is a water, and solute is the material of following final concentration: 10mMTris-HCl, the sodium laurylsulfonate of 0.5g/100ml (SDS), the NaCl aqueous solution of the EDTA of 1mM and 50mM, the Proteinase K of 0.5mg/ml.
Cracking is 6.4 in conjunction with liquid: pH, solvent is a water, solute is the Tris-HCl of the material of following final concentration: 50mM, the guanidinium isothiocyanate of 3M, the Triton X-100 of 1% (volume percent), 0.5g/100ml 3-(3-cholamine propyl group)-dimethylamino-1-propanesulfonic acid (CHAPS), the ethanol of the EDTA of 10mM and 15% (volumn concentration).
Washings I: solvent is a water, and solute is the Guanidinium hydrochloride of the material of following final concentration: 4M, the ethanol of 25% (volumn concentration) and the EDTA of 10mM.
Cleaning solution II: 75% (volumn concentration) aqueous ethanolic solution.
Elutriant: be TE solution, the pH of described TE solution is 8.0, and solvent is a water, and solute is the Tris-HCl of 10mM, the EDTA of 1mM.
Bead suspension: the nano silicone bag in every ml bead suspension is 50mg by magnetic microsphere (diameter is 60nm) (the micro-nano novel material Science and Technology Ltd. of moistening difficult to understand), and all the other are water.
2, the reagent with step 1 extracts purify DNA
1) pre-treatment
In the 1.5ml centrifuge tube, at first add the 100ul pre-treated lysis solution, then the liquid anticoagulation of 0.1ul is added in the pre-treated lysis solution, the vortex vibration, 56 ℃ of temperature are bathed 30min.
Fully after the vibration,, obtain thick lysate by centrifugal removal carrier (referring to the solid matter that biomass cells adheres to or adheres to).
2) nucleic acid is adsorbed in solid phase carrier
Add the cracking of bead suspension (add-on is that 10 μ l concentration are the solution of 50mg/ml) and 300 μ l respectively in conjunction with liquid in the thick lysate that obtains toward step 1, formed the complex body of magnetic Nano microsphere-DNA, this complex body is in the solution system that slightly lysate and cracking are formed in conjunction with liquid.
3) complex body separates
After the centrifuge tube vortex oscillation, be put on the magnet stand, inhale and to abandon liquid, this step is under the effect of extraneous magnetic field force, with step 2) in the complex body of magnetic Nano microsphere-DNA from the solution system that thick lysate and cracking are formed in conjunction with liquid, isolate.
4) washing
Add 500 μ l washings I in centrifuge tube, vortex oscillation washing back is inhaled and is abandoned liquid;
Add 500 μ l cleaning solution II in centrifuge tube, vortex oscillation washing back is inhaled and is abandoned liquid, repeats this operation once.
Washing can be removed the material outside the nucleic acid that is combined on the solid phase carrier (magnetic Nano microsphere).
5) wash-out
After washing finishes, blot residual liquid with 20ul rifle head, room temperature is dried 5-10min and (when room temperature surpasses 30 ℃, is dried 5min and get final product; Dry 7-10 minute when room temperature is lower than 30 ℃, but can not surpass 10min at most).The elutriant that adds 30 μ l then in the centrifuge tube, 65 ℃ of temperature are bathed 5-8min, and magnetic separates, and draws DNA, 4 ℃ or-20 ℃ of preservations.
3, the STR composite amplification is analyzed
The DNA that embodiment 1 is extracted carries out the STR composite amplification, and what composite amplification adopted is commercially available commercial STR composite amplification reagent kit (as the Id test kit of American AB I company).Detect with 3130 type genetic analyzers then, the result is shown in Figure 1A, the STR site is complete, somatotype is accurate, phenomenons such as no allelic loss, uneven amplification, Pullup peak, stutter band, split peak occur, show this purification process purification efficiency height, the DNA purity height that obtains, integrity is good.
Two, DNA and the concentration of extracting in the purifying micro blood detects
1, extracts DNA
From the liquid anticoagulation of different volumes (1,2,3,4 and 5 μ l), extract DNA.The method of extracting is identical with above-mentioned steps one, and its difference is the amount of agents useful for same:
The method of extracting the DNA in the liquid anticoagulation of 1,2,3,4 μ l is identical with above-mentioned steps one, and used pre-treated lysis solution all is 100 μ l; Bead suspension all is 10ul; Cracking all is 300ul in conjunction with liquid; Washings I all is 500ul; Cleaning solution II all is 500ul; Elutriant all is 30ul.
Extract the DNA in the liquid anticoagulation of 5 μ l, used pre-treated lysis solution 200 μ l; Bead suspension 20ul; Cracking is in conjunction with liquid 600ul; Washings I 500ul; Cleaning solution II 500ul; Elutriant 50ul.
Utilize real-time fluorescence quantitative PCR test kit (Quantifler
TMHuman, ABI company) and
The fluorescent quantitation system detects the concentration of the DNA that is extracted, and the result is as shown in table 1, the whole extraction efficiency height of this test kit.
Table 1 also shows: adopt two kinds of different quantivative approachs (real-time fluorescence quantitative PCR and dyestuff are quantitative), two kinds of method quantitative results are suitable, show DNA integrity brilliance, show that DNA dna break in the process of extracting is few.
The concentration detected result of the DNA that extracts in the liquid anticoagulation of table 1. different volumes
Three, DNA extraction efficiency rating
Adopt 300ng DNA standard substance (G1471, Promega company) as initial DNA (60ul), the reagent and the method that adopt embodiment 1 step 1 to provide are extracted, the DNA that detects final recovery by DNA fluorescent quantitation method measures, and calculates DNA extraction efficient according to formula [amounts (300ng) of the DNA yield (ng) that extraction efficiency=wash-out obtains/adding DNA standard substance].
Wherein, the experimental design of quantitative fluorescent PCR is as follows:
1) 7 parts of standard substance that contain 300ng DNA (30ul system) of preparation, wherein 6 parts as sample, adopts reagent of the present invention and method to extract.Remaining a as positive control, after DNA extraction, carry out electrophoresis relatively with all the other 6 parts of same standard product;
2) for the ease of comparing, 6 are extracted samples and still adopt 30ul system wash-out;
3) after obtaining 6 30ulDNA samples, finally get the 20ul sample equally in each 30 system, last sample also carries out electrophoresis detection.
(M is molecular weight Marker (λ/HindIII dna molecular amount Marker), its molecular weight ranges 500bp-23kb for electrophorogram such as Figure 1B; 1-6 extracts the DNA (getting the 20ul electrophoresis in the 30ul system) that obtains for this patent provides reagent and method; Positive control is for containing shown in the 300ng DNA standard substance (getting the 20ul electrophoresis in the 30ul system), electrophoresis result by 1-6 sample and positive control compares, find aspect and the positive control basically identicals such as brightness, width and position of 1-6 sample at the DNA electrophoretic band, show that the DNA loss is little in leaching process, DNA extraction efficient height, to extract the DNA integrity good.And DNA fluorescent quantitation result shows that reagent and method DNA extraction efficient that this invention provides are higher than 90%.
Quantitative result is as shown in table 2, and DNA extraction efficient is up to 93.67%.
Table 2.DNA extraction efficiency
DNA and compliance test result in embodiment 2, the extraction purifying blood cake
3 * 3mm blood cake on the FTA card as biological sample, is extracted the DNA in this sample of purifying.
One, the DNA in the extraction purifying blood cake
1, reagent
Reagent is as follows:
Pre-treated lysis solution: pH is 8.0, and solvent is a water, and solute is the material of following final concentration: 50mMTris-HCl, the sodium laurylsulfonate of 3g/100ml (SDS), the NaCl aqueous solution of the CDTA of 50mM and 100mM, the Proteinase K of 0.2mg/ml.
Cracking is 7.0 in conjunction with liquid: pH, solvent is a water, solute is the Tris-HCl of the material of following final concentration: 100mM, the guanidinium isothiocyanate of 5M, the Triton X-100 of 5% (volume percent), the 3-of 2g/100ml (3-cholamine propyl group)-dimethylamino-1-propanesulfonic acid (CHAPS), the ethanol of the EDTA of 50mM and 20% (volume percent).
Washings I: solvent is a water, and solute is the Guanidinium hydrochloride of the material of following final concentration: 5M, the ethanol of 35% (volumn concentration) and the EDTA of 50mM.
Cleaning solution II: 75% (volumn concentration) aqueous ethanolic solution.
Elutriant: be TE solution, the pH of described TE solution is 8.0, and solvent is a water, and solute is the Tris-HCl of 10mM, the EDTA of 1mM.
Bead suspension: the nano silicone bag in every ml bead suspension is 50mg by magnetic microsphere (diameter is 200nm) (the micro-nano novel material Science and Technology Ltd. of moistening difficult to understand), and all the other are water.
2, the reagent with step 1 extracts purify DNA
1) pre-treatment
In the 1.5ml centrifuge tube, at first add the 200ul pre-treated lysis solution, then 3 * 3mm blood cake on the FTA card is added in the pre-treated lysis solution, the vortex vibration, 56 ℃ of temperature are bathed 30min.
Fully after the vibration,, obtain thick lysate by centrifugal removal carrier (referring to the solid matter that biomass cells adheres to or adheres to).
2) nucleic acid is adsorbed in solid phase carrier
Add the cracking of bead suspension (add-on is that 20 μ l concentration are the solution of 50mg/ml) and 600 μ l respectively in conjunction with liquid in the thick lysate that obtains toward step 1, formed the complex body of magnetic Nano microsphere-DNA, this complex body is in the solution system that slightly lysate and cracking are formed in conjunction with liquid.
3) complex body separates
After the centrifuge tube vortex oscillation, be put on the magnet stand, inhale and to abandon liquid, this step is under the effect of extraneous magnetic field force, with step 2) in the complex body of magnetic Nano microsphere-DNA from the solution system that thick lysate and cracking are formed in conjunction with liquid, isolate.
4) washing
Add 500 μ l washings I in centrifuge tube, vortex oscillation washing back is inhaled and is abandoned liquid; Repeat this operation once
Add 500 μ l washings I in centrifuge tube, vortex oscillation washing back is inhaled and is abandoned liquid, repeats this operation once.
Washing can be removed the material outside the nucleic acid that is combined on the solid phase carrier (magnetic Nano microsphere).
5) wash-out
After washing finishes, blot residual liquid with 20ul rifle head, room temperature is dried 5-10min and (when room temperature surpasses 30 ℃, is dried 5min and get final product; Dry 7-10 minute when room temperature is lower than 30 ℃, but can not surpass 10min at most).The elutriant that adds 50 μ l then in the centrifuge tube, 65 ℃ of temperature are bathed 5-8min, and magnetic separates, and draws DNA, 4 ℃ or-20 ℃ of preservations.
Two, the STR composite amplification is analyzed
The DNA that above-mentioned steps is obtained carries out the analysis of STR composite amplification, its method is identical with the method that embodiment 1 provides, the result as shown in Figure 2, the STR site is complete, somatotype is accurate, and phenomenons such as no allelic loss, uneven amplification, Pull up peak, stutter band, split peak occur, and show this purification process purification efficiency height, the DNA purity height that obtains, integrity is good.
DNA and compliance test result in embodiment 3, the extraction purifying blood cake
2 μ l anticoagulations are dropped in sample on the cotton swab as biological sample, extract the DNA in this sample of purifying.
One, extracts purify DNA
1, reagent
Reagent is as follows:
Pre-treated lysis solution: pH is 8.5, and solvent is a water, and solute is the material of following final concentration: 100mM phosphoric acid salt (100mM NaH
2PO
4, 20mM NaCl, pH 8.2), the sarcosyl of 5.0g/100ml (SLS), the NaCl aqueous solution of the EGTA of 100mM and 150mM, the Proteinase K of 5.0mg/ml.
Cracking is 7.4 in conjunction with liquid: pH, solvent is a water, solute is the Tris-HCl of the material of following final concentration: 150mM, the guanidine thiocyanate of 8M, the Tween 20 of 10% (volume percent), 4.0g/100ml 3-(3-cholamine propyl group)-dimethylamino-1-propanesulfonic acid (CHAPS), the Virahol of the EGTA of 100mM and 30% (volumn concentration).
Washings I: solvent is a water, and solute is the guanidine thiocyanate of the material of following final concentration: 6M, the ethanol of 50% (volumn concentration) and the EDTA of 100mM.
Cleaning solution II: 80% (volumn concentration) aqueous ethanolic solution.
Elutriant: be TE solution, the pH of described TE solution is 8.0, and solvent is a water, and solute is the Tris-HCl of 20mM, the EDTA of 2mM.
Bead suspension: the nano silicone bag in every ml bead suspension is 50mg by magnetic microsphere (diameter is 600nm) (the micro-nano novel material Science and Technology Ltd. of moistening difficult to understand), and all the other are water.
2, the reagent with step 1 extracts purify DNA
1) pre-treatment
At first add the 200ul pre-treated lysis solution in the 1.5ml centrifuge tube, the liquid anticoagulation of 2ul that will drop in then on the cotton swab adds in the pre-treated lysis solution, the vortex vibration, and 56 ℃ of temperature are bathed 30min.
Fully after the vibration,, obtain thick lysate by centrifugal removal carrier (referring to the solid matter that biomass cells adheres to or adheres to).
2) nucleic acid is adsorbed in solid phase carrier
Add the cracking of bead suspension (add-on is that 20 μ l concentration are the solution of 50mg/ml) and 600 μ l respectively in conjunction with liquid in the thick lysate that obtains toward step 1), formed the complex body of magnetic Nano microsphere-DNA, this complex body is in the solution system that slightly lysate and cracking are formed in conjunction with liquid.
3) complex body separates
After the centrifuge tube vortex oscillation, be put on the magnet stand, inhale and to abandon liquid, this step is under the effect of extraneous magnetic field force, with step 2) in the complex body of magnetic Nano microsphere-DNA from the solution system that thick lysate and cracking are formed in conjunction with liquid, isolate.
4) washing
Add 500 μ l washings I in centrifuge tube, vortex oscillation washing back is inhaled and is abandoned liquid; Repeat this operation once
Add 500 μ l cleaning solution II in centrifuge tube, vortex oscillation washing back is inhaled and is abandoned liquid, repeats this operation once.
Washing can be removed the material outside the nucleic acid that is combined on the solid phase carrier (magnetic Nano microsphere).
5) wash-out
After washing finishes, blot residual liquid with 20ul rifle head, room temperature is dried 5-10min and (when room temperature surpasses 30 ℃, is dried 5min and get final product; Dry 7-10 minute when room temperature is lower than 30 ℃, but can not surpass 10min at most).The elutriant that adds 50 μ l then in the centrifuge tube, 65 ℃ of temperature are bathed 5-8min, and magnetic separates, and draws DNA, 4 ℃ or-20 ℃ of preservations.
Two, the STR composite amplification is analyzed
The DNA that above-mentioned steps is obtained carries out the analysis of STR composite amplification, its method is identical with the method that embodiment 1 provides, the result as shown in Figure 3, the STR site is complete, somatotype is accurate, and phenomenons such as no allelic loss, uneven amplification, Pull up peak, stutter band, split peak occur, and show that this purification process can effectively extract the DNA in the carrier surface institute adherent cell, the DNA purity height that obtains, integrity is good.
DNA and compliance test result in embodiment 4, the extraction purifying blood cake
2 μ l anticoagulations, PCR inhibitor are dropped in the blood cake that forms on the cotton as biological sample, extract the DNA in this sample of purifying, wherein, this inhibitor is the mixture of 1ul protoheme (0.5mM) and 1ul humic acid (2.5mg/ml) composition.
One, extracts purify DNA
1, reagent
Reagent is as follows:
Pre-treated lysis solution: pH is 7.7, and solvent is a water, and solute is the material of following final concentration: 30mMTris-HCl, the sarcosyl of 2.0g/100ml (SLS), the NaCl aqueous solution of the EGTA of 30mM and 80mM, the Proteinase K of 2.0mg/ml.
Cracking is 7.1 in conjunction with liquid: pH, solvent is a water, solute is the Tris-HCl of the material of following final concentration: 80mM, the guanidine thiocyanate of 4M, the Tween 20 of 2% (volume percent), 1.5g/100ml 3-(3-cholamine propyl group)-dimethylamino-1-propanesulfonic acid (CHAPS), the Virahol of the EGTA of 30mM and 24% (volumn concentration).
Washings I: solvent is a water, and solute is the Guanidinium hydrochloride of the material of following final concentration: 4.4M, the ethanol of 30% (volumn concentration) and the EDTA of 30mM.
Cleaning solution II: 80% (volumn concentration) aqueous ethanolic solution.
Elutriant: be TE solution, the pH of described TE solution is 8.0, and solvent is a water, and solute is the EDTA of 1mM.
Bead suspension: the nano silicone bag in every ml bead suspension is 50mg by magnetic microsphere (diameter is 1000nm) (the micro-nano novel material Science and Technology Ltd. of moistening difficult to understand), and all the other are water.
2, the reagent with step 1 extracts purify DNA
1) pre-treatment
At first add the 200ul pre-treated lysis solution in the 1.5ml centrifuge tube, the biological sample with present embodiment adds in the pre-treated lysis solution then, the vortex vibration, and 56 ℃ of temperature are bathed 30min.
Fully after the vibration,, obtain thick lysate by centrifugal removal carrier (referring to the solid matter that biomass cells adheres to or adheres to).
2) nucleic acid is adsorbed in solid phase carrier
Add the cracking of bead suspension (add-on is that 20 μ l concentration are the solution of 50mg/ml) and 600 μ l respectively in conjunction with liquid in the thick lysate that obtains toward step 1, formed the complex body of magnetic Nano microsphere-DNA, this complex body is in the solution system that slightly lysate and cracking are formed in conjunction with liquid.
3) complex body separates
After the centrifuge tube vortex oscillation, be put on the magnet stand, inhale and to abandon liquid, this step is under the effect of extraneous magnetic field force, with step 2) in the complex body of magnetic Nano microsphere-DNA from the solution system that thick lysate and cracking are formed in conjunction with liquid, isolate.
4) washing
Add 500 μ l washings I in centrifuge tube, vortex oscillation washing back is inhaled and is abandoned liquid; Repeat this operation once
Add 500 μ l cleaning solution II in centrifuge tube, vortex oscillation washing back is inhaled and is abandoned liquid, repeats this operation once.
Washing can be removed the material outside the nucleic acid that is combined on the solid phase carrier (magnetic Nano microsphere).
5) wash-out
After washing finishes, blot residual liquid with 20ul rifle head, room temperature is dried 5-10min and (when room temperature surpasses 30 ℃, is dried 5min and get final product; Dry 7-10 minute when room temperature is lower than 30 ℃, but can not surpass 10min at most).The elutriant that adds 50 μ l then in the centrifuge tube, 65 ℃ of temperature are bathed 5-8min, and magnetic separates, and draws DNA, 4 ℃ or-20 ℃ of preservations.
Two, the STR composite amplification is analyzed
The DNA that above-mentioned steps is obtained carries out the analysis of STR composite amplification, its method is identical with the method that embodiment 1 provides, the result as shown in Figure 4, STR peak type is better, and peak value is higher, and the STR site is complete, somatotype is accurate, phenomenon appearance such as no allelic loss, uneven amplification, Pull up peak, stutter band, split peak show this purification process purification efficiency height, and the DNA concentration height that obtains, purity is tool integrity preferably also well.
DNA and compliance test result in embodiment 5, the extraction purifying blood cake
2 μ l anticoagulations are dropped on the jeans as biological sample, extract the DNA in this sample of purifying.
One, extracts purify DNA
1, reagent
Reagent is as follows:
Pre-treated lysis solution: pH is 8.3, and solvent is a water, and solute is the material of following final concentration: 70mMTris-HCl, the sarcosyl of 4g/100ml (SLS), the NaCl aqueous solution of the EGTA of 70mM and 120mM, the Proteinase K of 4mg/ml.
Cracking is 7.3 in conjunction with liquid: pH, solvent is a water, solute is the Tris-HCl of the material of following final concentration: 120mM, the Guanidinium hydrochloride of 6M, the Tween 20 of 3% (volumn concentration), the 3-of 3g/100ml (3-cholamine propyl group)-dimethylamino-1-propanesulfonic acid (CHAPS), the polyoxyethylene glycol of the EGTA of 70mM and 26% (volumn concentration).
Washings I: solvent is a water, and solute is the potassiumiodide of the material of following final concentration: 5.6M, the ethanol of 45% (volumn concentration) and the EDTA of 70mM.
Cleaning solution II: 75% (volumn concentration) aqueous ethanolic solution.
Elutriant: be TE solution, the pH of described TE solution is 8.0, and solvent is a water, and solute is the Tris-HCl of 10mM.
Bead suspension: the nano silicone bag in every ml bead suspension is 50mg by magnetic microsphere (diameter is 1200nm) (the micro-nano novel material Science and Technology Ltd. of moistening difficult to understand), and all the other are water.
2, the reagent with step 1 extracts purify DNA
It is different that the difference of the method that the method for extracting purify DNA and embodiment 3 provide is to extract the reagent of purify DNA, and all the other are identical.
Two, the STR composite amplification is analyzed
The DNA that above-mentioned steps is obtained carries out the analysis of STR composite amplification, its method is identical with the method that embodiment 1 provides, the result as shown in Figure 5, the STR site is complete, somatotype is accurate, phenomenon appearance such as no allelic loss, uneven amplification, Pull up peak, stutter band, split peak show this purification process purification efficiency height, and the DNA concentration height that obtains, purity is tool integrity preferably also well.
DNA and compliance test result in embodiment 6, the extraction purifying seminal stain
1 μ l seminal fluid is dropped on the colourless cotton as biological sample, extract the DNA in this sample of purifying.
There are fine difference in the extraction of seminal stain and conventional sample, and promptly needing to add DTT (dithiothreitol (DTT)) in pre-treated lysis solution could normal extraction.
One, extracts purify DNA
1, reagent
Reagent is as follows:
Pre-treated lysis solution: pH is 7.6, and solvent is a water, and solute is the material of following final concentration: 90mMTris-HCl, 4.5g/100ml sodium laurylsulfonate (SDS), the NaCl aqueous solution of the EDTA of 6mM and 70mM, the Proteinase K of 1.0mg/ml, 0.05M DTT (dithiothreitol (DTT)).
Cracking is 7.2 in conjunction with liquid: pH, solvent is a water, solute is the Tris-HCl of the material of following final concentration: 20mM, the guanidine thiocyanate of 7M, the Tween 21 of 2.5% (volume percent), 1.0g/100ml 3-(3-cholamine propyl group)-dimethylamino-1-propanesulfonic acid (CHAPS), the Virahol of the EGTA of 20mM and 18% (volumn concentration).
Washings I: solvent is a water, and solute is the sodium perchlorate of the material of following final concentration: 4.6M, the ethanol of 40% (volumn concentration) and the EDTA of 15mM.
Cleaning solution II: 75% aqueous ethanolic solution.
Elutriant: be TE solution, the pH of described TE solution is 8.0, and solvent is a water, and solute is the Tris-HCl of 10mM, the EDTA of 1mM.
Bead suspension: the nano silicone bag in every ml bead suspension is 100mg by magnetic microsphere (diameter is 50nm) (the micro-nano novel material Science and Technology Ltd. of moistening difficult to understand), and all the other are water.
2, the reagent with step 1 extracts purify DNA
1) pre-treatment
At first add the 200ul pre-treated lysis solution in the 1.5ml centrifuge tube, the biological sample with present embodiment adds in the pre-treated lysis solution then, the vortex vibration, and 56 ℃ of temperature are bathed 30min.
Fully after the vibration,, obtain thick lysate by centrifugal removal carrier (referring to the solid matter that biomass cells adheres to or adheres to).
2) nucleic acid is adsorbed in solid phase carrier
Add the cracking of bead suspension (add-on is that 20 μ l concentration are the solution of 100mg/ml) and 600 μ l respectively in conjunction with liquid in the thick lysate that obtains toward step 1), formed the complex body of magnetic Nano microsphere-DNA, this complex body is in the solution system that slightly lysate and cracking are formed in conjunction with liquid.
3) complex body separates
After the centrifuge tube vortex oscillation, be put on the magnet stand, inhale and to abandon liquid, this step is under the effect of extraneous magnetic field force, with step 2) in the complex body of magnetic Nano microsphere-DNA from the solution system that thick lysate and cracking are formed in conjunction with liquid, isolate.
4) washing
Add 500 μ l washings I in centrifuge tube, vortex oscillation washing back is inhaled and is abandoned liquid; Repeat this operation once
Add 500 μ l cleaning solution II in centrifuge tube, vortex oscillation washing back is inhaled and is abandoned liquid, repeats this operation once.
Washing can be removed the material outside the nucleic acid that is combined on the solid phase carrier (magnetic Nano microsphere).
5) wash-out
After washing finishes, blot residual liquid with 20ul rifle head, room temperature is dried 5-10min and (when room temperature surpasses 30 ℃, is dried 5min and get final product; Dry 7-10 minute when room temperature is lower than 30 ℃, but can not surpass 10min at most).The elutriant that adds 50 μ l then in the centrifuge tube, 65 ℃ of temperature are bathed 5-8min, and magnetic separates, and draws DNA, 4 ℃ or-20 ℃ of preservations.
Two, the STR composite amplification is analyzed
The DNA that above-mentioned steps is obtained carries out the analysis of STR composite amplification, its method is identical with the method that embodiment 1 provides, the result as shown in Figure 6, the STR collection of illustrative plates shows that the STR peak value is higher, the STR site is complete, and somatotype is accurate, and phenomenons such as no allelic loss, uneven amplification, Pull up peak, stutter band, split peak occur, show this purification process purification efficiency height, the DNA concentration height that obtains, purity are well and tool integrity preferably.
DNA and compliance test result in embodiment 7, the extraction purified salivary
50 μ l salivas are dropped in the buccal swab that forms on the cotton swab as biological sample, extract the DNA in this sample of purifying.
One, extracts purify DNA
1, reagent
The difference of agents useful for same and embodiment 2 is that the concentration of described sodium chloride aqueous solution is 130mM, and all the other reagent are identical.
2, the reagent with step 1 extracts purify DNA
The amount of extracting the step of purify DNA and step that embodiment 2 provides and used each reagent with the reagent of step 1 is identical.
Two, the STR composite amplification is analyzed
The DNA that above-mentioned steps is obtained carries out the analysis of STR composite amplification, its method is identical with the method that embodiment 1 provides, the result as shown in Figure 7, the STR collection of illustrative plates shows that the STR peak value is higher, the STR site is complete, and somatotype is accurate, and phenomenons such as no allelic loss, uneven amplification, Pull up peak, stutter band, split peak occur, show this purification process purification efficiency height, the DNA concentration height that obtains, purity are well and tool integrity preferably.
DNA and compliance test result in embodiment 8, the extraction purifying stub
Stub as biological sample, is extracted the DNA in this sample of purifying.
One, extracts purify DNA
1, reagent
The difference of agents useful for same and embodiment 7 is that the concentration of used sodium chloride solution is 90mM, and all the other reagent are identical.
2, the reagent with step 1 extracts purify DNA
Extracting method is identical with embodiment 7.
Two, the STR composite amplification is analyzed
The DNA that above-mentioned steps is obtained carries out the analysis of STR composite amplification, its method is identical with the method that embodiment 1 provides, the result as shown in Figure 8, the STR collection of illustrative plates shows that the STR peak value is higher, the STR site is complete, and somatotype is accurate, and phenomenons such as no allelic loss, uneven amplification, Pull up peak, stutter band, split peak occur, show this purification process purification efficiency height, the DNA concentration height that obtains, purity are well and tool integrity preferably.
DNA and compliance test result in embodiment 9, the extraction purifying hair
The hair root part (total length is about 1-3mm) of the hair that comes off naturally as biological sample, is extracted the DNA in this sample of purifying.
The extraction of hair and conventional sample also there are differences, and promptly needing to add DTT (dithiothreitol (DTT)) in pre-treated lysis solution could normal extraction.
One, extracts purify DNA
1, reagent
The difference of agents useful for same and embodiment 6 is that the concentration of used sodium chloride solution is 110mM, and all the other reagent are identical.
2, the reagent with step 1 extracts purify DNA
Extraction step is identical with embodiment 6.
Two, the STR composite amplification is analyzed
The DNA that above-mentioned steps is obtained carries out the analysis of STR composite amplification, its method is identical with the method that embodiment 1 provides, the result as shown in Figure 9, the STR site is complete, somatotype is accurate, phenomenon appearance such as no allelic loss, uneven amplification, Pull up peak, stutter band, split peak show this purification process purification efficiency height, and the DNA concentration height that obtains, purity is tool integrity preferably also well.
DNA and compliance test result in embodiment 10, the extraction tissue
With tissue piece (1-2mm
3Muscle tissue), extract the DNA in this sample of purifying as biological sample.
One, extracts purify DNA
1, reagent
The difference of agents useful for same and embodiment 7 is that the concentration of used sodium chloride solution is 140mM, and all the other reagent are identical.
2, the reagent with step 1 extracts purify DNA
Extraction step is identical with embodiment 7.
Two, the STR composite amplification is analyzed
The DNA that above-mentioned steps is obtained carries out the analysis of STR composite amplification, its method is identical with the method that embodiment 1 provides, the result as shown in figure 10, the STR site is complete, somatotype is accurate, phenomenon appearance such as no allelic loss, uneven amplification, Pull up peak, stutter band, split peak show this purification process purification efficiency height, and the DNA concentration height that obtains, purity is tool integrity preferably also well.
DNA and compliance test result in embodiment 11, the extraction human body cast-off cells
Cast-off cells on the mobile phone as biological sample, are extracted the DNA in this sample of purifying.Wherein, the obtaining step of cast-off cells may further comprise the steps: get a little cotton swab (10-20mg) with tweezers and soak back wiping cast-off cells place body surface, get a little dry cotton swab (10-20mg) subsequently and repeat the wiping wetted surface, two carriers that are stained with cast-off cells are joined in the pre-treated lysis solution in the lump the vortex vibration.
One, extracts purify DNA
1, reagent
Agents useful for same is identical with embodiment 1.
2, the reagent with step 1 extracts purify DNA
Extraction step is identical with embodiment 2.
Two, the STR composite amplification is analyzed
The DNA that above-mentioned steps is obtained carries out the analysis of STR composite amplification, its method is identical with the method that embodiment 1 provides, the result as shown in figure 11, the STR peak value is higher, the STR site is complete, and somatotype is accurate, and phenomenons such as no allelic loss, uneven amplification, Pull up peak, stutter band, split peak occur, show this purification process purification efficiency height, the DNA concentration height that obtains, purity are well and tool integrity preferably.
Embodiment 12-15
That embodiment 12 extracts is the DNA of the cast-off cells on the sweater, and that embodiment 13 extracts is the DNA of cast-off cells on the wallet, and that embodiment 14 extracts is the DNA of rim of a cup cast-off cells, and that embodiment 15 extracts is the DNA of keyboard cast-off cells.
The preparation method of the cast-off cells among the embodiment 12-15, the method for extracting used reagent of the DNA of these cast-off cells and extraction are all identical with embodiment 11, the result is shown in Figure 12,13,14 and 15 for its STR composite amplification analyzing and testing, the STR peak value is higher, the STR site is complete, somatotype is accurate, phenomenons such as no allelic loss, uneven amplification, Pull up peak, stutter band, split peak occur, show this purification process purification efficiency height, the DNA concentration height that obtains, purity are well and tool integrity preferably.
The corrupt blood cake of 3 * 3mm as biological sample, is extracted the DNA in this sample of purifying.
One, extracts purify DNA
1, reagent
Agents useful for same is identical with embodiment 1.
2, the reagent with step 1 extracts purify DNA
Extraction step is identical with embodiment 2.
Two, the STR composite amplification is analyzed
The DNA that above-mentioned steps is obtained carries out the analysis of STR composite amplification, its method is identical with the method that embodiment 1 provides, the result as shown in figure 16, the STR site is complete, do not have big fragment bit point and lose phenomenon, somatotype is accurate, and phenomenons such as no allelic loss, Pull up peak, stutter band, split peak occur, show this purification process purification efficiency height, obtained to meet the template DNA of STR composite amplification requirement
Embodiment 17, corrupt biological sample DNA extraction result
With 1-2mm
3Corrupt tissue as biological sample, extract the DNA in this sample of purifying.
One, extracts purify DNA
1, reagent
Agents useful for same is identical with embodiment 1.
2, the reagent with step 1 extracts purify DNA
Extraction step is identical with embodiment 2.
Two, the STR composite amplification is analyzed
The DNA that above-mentioned steps is obtained carries out the analysis of STR composite amplification, its method is identical with the method that embodiment 1 provides, the result as shown in figure 17, the STR site is complete, does not have big fragment STR site and loses, and somatotype is accurate, phenomenons such as no Pullup peak, stutter band, split peak occur, show this purification process purification efficiency height, gained DNA purity height, this reagent can obtain complete relatively dna fragmentation by purifying in corrupt biological specimen.
Claims (13)
1. reagent that extracts purify DNA, form by following material:
Pre-treated lysis solution: pH is 7.4-8.5, solvent is a water, and solute is the material of following final concentration: 10-100mM buffering salt, the anion surfactant of 0.5-5g/100ml, the sequestrant of 1-100mM, the soluble salt of 50-150mM and the Proteinase K of 0.2-4mg/ml;
Cracking is in conjunction with liquid: pH=6.4-7.4, solvent is a water, solute is the buffering salt of the material of following final concentration: 20-150mM, the Chaotropic salt of 3-8M, the nonionogenic tenside of 1-10% (volume percent), 0.5-4g/100ml zwitterionic detergent, the alcohol of the sequestrant of 10-100mM and 15-30% (volumn concentration);
Washings I: solvent is a water, and solute is the Chaotropic salt of the material of following final concentration: 4-6M, the sequestrant of the pure and mild 10-100mM of 25-50% (volumn concentration);
Cleaning solution II: 75-80% (volumn concentration) aqueous ethanolic solution;
Elutriant: be TE solution, the pH of described TE solution is 8.0, and solvent is a water, and solute is the Tris-HCl of 0-20mM, the EDTA of 0-2mM;
Bead suspension: the composition of every ml bead suspension is as follows: diameter be the nano silicone bag of 50-1200nm by magnetic microsphere 50-100mg, all the other are water.
2. reagent that extracts purify DNA, form by following material:
Pre-treated lysis solution: pH is 7.4-8.5, solvent is a water, solute is the material of following final concentration: the 10-100mM buffering salt, 0.5-5g/100ml anion surfactant, the sequestrant of 1-100mM, the dithiothreitol (DTT) of the soluble salt of 50-150mM and the Proteinase K of 0.2-4mg/ml and 0.025-0.05M;
Cracking is in conjunction with liquid: pH=6.4-7.4, solvent is a water, solute is the buffering salt of the material of following final concentration: 20-150mM, the Chaotropic salt of 3-8M, the nonionogenic tenside of 1-10% (volume percent), 0.5-4g/100ml zwitterionic detergent, the alcohol of the sequestrant of 10-100mM and 15-30% (volumn concentration);
Washings I: solvent is a water, and solute is the Chaotropic salt of the material of following final concentration: 4-6M, the sequestrant of the pure and mild 10-100mM of 25-50% (volumn concentration);
Cleaning solution II: 75-80% (volumn concentration) aqueous ethanolic solution;
Elutriant: be TE solution, the pH of described TE solution is 8.0, and solvent is a water, and solute is the Tris-HCl of 0-20mM, the EDTA of 0-2mM;
Bead suspension: the composition of every ml bead suspension is as follows: diameter be the nano silicone bag of 50-1200nm by magnetic microsphere 50-100mg, all the other are water.
3. reagent according to claim 1 and 2, it is characterized in that: in the described pre-treated lysis solution, the concentration of buffering salt is 20-80mM, the concentration of anion surfactant is 0.5-4g/100ml, the concentration of sequestrant is 20-80mM, the concentration of soluble salt is 50-130mM, and the concentration of Proteinase K is 0.2-2mg/ml; Described anion surfactant is sodium laurylsulfonate or sarcosyl; Described soluble salt solution is the NaCl aqueous solution.
4. reagent according to claim 3 is characterized in that: the concentration of described soluble salt is 80-100mM; The concentration of Proteinase K is 0.2-1mg/ml.
5. reagent according to claim 1 and 2, it is characterized in that: described cracking is in conjunction with in the liquid, the concentration of Chaotropic salt is 4-6M, the concentration of buffering salt is 50-120mM, the concentration of sequestrant is 20-80mM, the concentration of nonionogenic tenside is 2-8% (volume percent), and the concentration of zwitterionic detergent is 1-3g/100ml, and the concentration of alcohol is 20-28% (volumn concentration); Described nonionogenic tenside is Triton X-100, Tween20, Tween 21, Tween.40, Tween 60, Tween 80, Tween 85, NP-40, Brij 30 or Span 20; Described zwitterionic detergent is 3-(3-cholamine propyl group)-dimethylamino-1-propanesulfonic acid; Described alcohol is ethanol, Virahol or polyoxyethylene glycol.
6. reagent according to claim 3, it is characterized in that: described cracking is in conjunction with in the liquid, the concentration of Chaotropic salt is 4-6M, the concentration of buffering salt is 50-120mM, the concentration of sequestrant is 20-80mM, the concentration of nonionogenic tenside is 2-8% (volume percent), and the concentration of zwitterionic detergent is 1-3g/100ml, and the concentration of alcohol is 20-28% (volumn concentration); Described nonionogenic tenside is Triton X-100, Tween 20, Tween 21, Tween.40, Tween 60, Tween 80, Tween 85, NP-40, Brij 30 or Span20; Described zwitterionic detergent is 3-(3-cholamine propyl group)-dimethylamino-1-propanesulfonic acid; Described alcohol is ethanol, Virahol or polyoxyethylene glycol.
7. reagent according to claim 1 and 2 is characterized in that: among the described washings I, the concentration of Chaotropic salt is 4.2-5.8M, and alcohol is the ethanol of 30-45% (volumn concentration), and the concentration of sequestrant is 20-80mM.
8. reagent according to claim 1 and 2 is characterized in that: described cleaning solution II is 75% (volume percent) aqueous ethanolic solution.
9. reagent according to claim 1 and 2 is characterized in that: the concentration of the Tris-HCl in the described elutriant is that the concentration of 10mM, EDTA is 1mM, and described elutriant pH is 8.0.
10. reagent according to claim 1 and 2 is characterized in that: the nano silicone bag in described every ml bead suspension is 50mg by magnetic microsphere.
11. reagent according to claim 1 and 2 is characterized in that: described buffering salt is Tris-HCl, phosphoric acid salt or HEPES salt; Described sequestrant is Citrate trianion, EDTA, EGTA or CDTA; Described Chaotropic salt is guanidinium isothiocyanate, guanidine thiocyanate, Guanidinium hydrochloride, urea, sodium iodide, potassiumiodide, lithium chloride, sodium perchlorate or Trichloroacetate.
12. reagent according to claim 11 is characterized in that: described Chaotropic salt is guanidinium isothiocyanate, guanidine thiocyanate or Guanidinium hydrochloride.
13. the test kit that the arbitrary described reagent of claim 1-12 is made.
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| CN102796727B (en) * | 2011-05-24 | 2014-06-18 | 博奥生物有限公司 | Method for extracting nucleic acid of gram positive bacteria |
| CN103031300B (en) * | 2011-10-09 | 2016-08-24 | 嘉和生物药业有限公司 | A kind of paramagnetic particle method extracting residual DNA from recombiant protein goods |
| CN102827831A (en) * | 2012-08-31 | 2012-12-19 | 武汉百泰基因工程有限公司 | Nucleic acid releasing reagent and nucleic acid releasing method |
| CN102888397A (en) * | 2012-09-25 | 2013-01-23 | 杭州硕航生物科技有限公司 | Kit using magnetic bead to extract whole blood genomic DNA and use of kit |
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