CN109280662A - One kind being based on silicon class medium microsphere ultramicron DNA extraction purification kit - Google Patents
One kind being based on silicon class medium microsphere ultramicron DNA extraction purification kit Download PDFInfo
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Abstract
The invention discloses one kind to be based on silicon class medium microsphere ultramicron DNA extraction purification kit, it include four cracking, absorption, rinsing, elution parts, cracking section is optimized mainly for except sample part, pyrolysis product is obtained to the maximum extent while facilitating operation, designs centrifuge tube and the matched hand basket with filtering function；The absorbability enhancer of absorbed portion increases hydrophobic effect, eliminates pipe wall electrostatic effect, promotes DNA and the effect of silicon class medium microsphere surface group, so that adsorption efficiency greatly improves；The efficient eluent of elution fraction innovatively uses competitive elution principle, can significantly improve DNA elution efficiency, reduces the residual of silicon class medium microsphere surface DNA.This case is improved to be based on silicon class medium microsphere ultramicron DNA extraction purification kit, easy to operate, the DNA yield of extraction is high, purity is high, complete fragment, in summary feature is especially suitable for the extraction of ultramicron biological material DNA, meets the high detection rate requirement increasingly proposed.
Description
Technical field
The present invention relates to a kind of kits, and in particular to one kind is tried based on silicon class medium microsphere ultramicron DNA extraction purification
Agent box.Belong to technical field of molecular biology.
Background technique
DNA extraction purification is very important link in molecular Biological Detection, the yield and purity direct relation of nucleic acid
To detected downstream success.Common nucleic acid purification method generally all includes substance with nucleic acid ining conjunction with of cell cracking, removal, sinks
Impurity, the purifies and separates DNA such as shallow lake nucleic acid, removal salt, organic solvent and etc..
Common method is mainly organic method, microballon absorption method, Chelex-100 method and centrifugal column method, and organic method step is numerous
It is trivial, time-consuming, and the big organic solvent of the toxicity such as chloroform, phenol is used, there is certain harm to operator's health；
Chelex-100 uses liquid phase Purification: Principles, and after being cracked using heating cells, impurity is in chelating agent removal biological sample to reach
It is easy to operate to the purpose of purifying, but extracted DNA purity is inadequate, is not able to satisfy ultramicron biological sample DNA purifying
It is required that；Though centrifugal column method does not use organic solvent, DNA yield is not high, the not reproducible use of centrifugal column, extracts single sample valence
Lattice are expensive.
Using the extraction and separation technology of microballon absorption method, the reagent big without toxicity such as Chloroform Exposed, phenol and operation letter
Just, the short time can be achieved with quick, high quality the extraction of sample, is the important development direction of nucleic acid extraction, mentions with tradition
Take the advantage that method is incomparable.When nucleic acid is in high concentration chaotropic agent and low ph solution, it can specifically be adsorbed onto silicon
Class dielectric surface, and disintegrated down in less salt high pH solution, to realize that DNA is extracted.Wherein magnetic bead is due to having in magnetism
Core often uses on a workstation, realizes that the automation of DNA is extracted, but be directed to trace, ultramicron sample DNA extraction efficiency is low, sample
This detection success rate is less optimistic.Silica bead extraction method is not due to having magnetic core, so can not evade centrifugation step, exactly this
Point automatically extracts work station there is presently no a silica bead, but silica bead medium has many advantages compared to magnetic bead medium, is embodied in
Grain size, adsorption specific surface area, homogeneity；Still further aspect is then the compound that DNA and silica bead are formed in silica bead extraction process
To be lacked very much, in addition in terms of handling some dirtier samples by being collected by centrifugation to compare the magnetic collection that paramagnetic particle method uses and lose
Have great advantage compared to paramagnetic particle method, largely improves DNA yield.
Traditional silica bead method extracts DNA method, mainly there is following disadvantage:
1, cracking operation is cumbersome, and sample is put into centrifuge tube, and heating cracking after suitable lysate is added, and cracking terminates
Pyrolysis product is sucked out afterwards and is transferred to new centrifuge tube or is taken out sample with tweezers, these steps are troublesome, especially work as sample
Error is easy when measuring larger.
2, pyrolysis product loss is big, and pyrolysis product is either transferred to new pipe or is taken sample with tweezers by conventional method
Sample itself can be all lost out and glues the pyrolysis product inhaled, because cell has ruptured at this time, lose pyrolysis product meaning at this time
The direct losses of DNA.
3, lysis efficiency is not high, guanidinium isothiocyanate is contained in traditional silica bead method lysate, guanidine salt is as a kind of denaturant, energy
The activity of enough protease inhibition K to a certain extent, directly affects lytic effect.
4, adsorption efficiency is not high, and the silica bead adsorption of DNA of traditional silica bead method relies primarily on guanidinium isothiocyanate and buffer salt provides
With high salt and low ph conditions, adsorption efficiency finds afterwards less than 60% after measured.
5, elution efficiency is inadequate, traditional silica bead method elute it is more common be pH 8.0 TE or water, only rely on high pH and low
Salt environment carrys out eluted dna, and experiment discovery significant portion DNA still remains on silica bead, so the silica bead of some producers extracts examination
Agent box advocate band pearl expand, but with pearl amplification the drawbacks of it is apparent.
Summary of the invention
It is a kind of based on silicon class medium microsphere ultramicron the purpose of the present invention is to overcome above-mentioned the deficiencies in the prior art, providing
DNA extraction purification kit.
To achieve the above object, the present invention adopts the following technical solutions:
One kind being based on silicon class medium microsphere ultramicron DNA extraction purification kit, including cracking section, absorbed portion, drift
Part and elution fraction are washed, the cracking section includes centrifuge tube and matched hand basket, is equipped with filter membrane, the filter in the bottom of hand basket
Film stops moisture penetration (to guarantee that lysate will not penetrate into centrifugation when sample heating cracking in the case where not being centrifuged by tension
Guan Zhong, after having cracked under the action of the centrifugal force, liquid can be passed through under filter membrane into centrifuge tube, while filter membrane also plays
A degree of filtering and impurity removing function effectively blocks particle or cotton-shaped impurity in sample, but does not influence the logical of target dna molecule
It crosses, centrifugation dead angle is small, can effectively drain the lysate of the samples such as cotton swab absorption, realizes that farthest obtaining cracking produces
Object)；The absorbed portion includes combination buffer, silicon class medium microsphere suspension, absorbability enhancer, the table of silicon class medium microsphere
Silicone hydroxyl is at least contained in face, and the solvent of absorbability enhancer is water, and solute includes: 3~10% proton of mass concentration or non-proton molten
Agent, the DNA short-movie section amplified production of 80~120 μ g/mL external sources addition；The elution fraction includes efficient eluent, is efficiently washed
The solvent of de- liquid is water, and solute includes: the Tris-HCl of 5~20mM, 0.5~2mM chelating agent, 0.1~5mM multivalent anions,
PH 8.0~9.0.
Preferably, the hand basket is embedded in centrifuge tube, and hand basket and centrifuge tube share lid, and good leak tightness is mixed by inversion
No leakage.
It is further preferred that the hand basket is equipped with gas pressure relief slot, eliminate in cracking heating process since air pressure expansion causes to rush
It uncaps the hidden danger of sub- generation cross contamination.
Preferably, the cracking section further includes lysate, and the solvent of lysate is water, and solute includes: 5~50mM's
The chlorination of Tris-HCl, 0.5~3wt% surfactant, 0.5~20mM chelating agent, 0.5~1.5M guanidine hydrochloride, 50~120mM
The Proteinase K of sodium and 0.5~3mg/mL, pH 7.2~8.5.
It is further preferred that the surfactant be ionic surfactant or nonionic surfactant, more into
One step is preferably nonionic surfactant, is still more preferably polysorbas20 (TWEEN-20), polysorbate40 (TWEEN-40), spits
Temperature 80 (TWEEN-80), Triton X-100, NP40 or Span20, are still more preferably Triton X-100.It has been generally acknowledged that
Anionic surfactant such as SDS, SLS meeting and protein during heating, which combine, forms cigar shape compound, such object
Matter shows negative electrical charge, can influence the adsorption efficiency of target dna molecule with DNA competitive binding to silicon class medium microsphere surface.Sun
Ionic surface active agent can be combined directly with DNA, influenced it and be adsorbed to silicon class medium microsphere surface.
Preferably, the solvent of the combination buffer is water, and solute includes: the Tris-HCl of 40~150mM, 1~5wt%
The alcohol content of surfactant, the chaotropic agent of 5~6M, 10~50mM chelating agent and volumetric concentration 20~35%, pH 5.2~
6.0。
It is further preferred that the surfactant be ionic surfactant or nonionic surfactant, more into
One step is preferably nonionic surfactant, is still more preferably polysorbas20 (TWEEN-20), polysorbate40 (TWEEN-40), spits
Temperature 80 (TWEEN-80), Triton X-100, NP40 or Span20, are still more preferably Triton X-100.It has been generally acknowledged that
Anionic surfactant such as SDS, SLS meeting and protein during heating, which combine, forms cigar shape compound, such object
Matter shows negative electrical charge, can influence the adsorption efficiency of target dna molecule with DNA competitive binding to silicon class medium microsphere surface.Sun
Ionic surface active agent can be combined directly with DNA, influenced it and be adsorbed to silicon class medium microsphere surface.
It is further preferred that the alcohol is any one of ethyl alcohol, methanol or isopropanol, it is still more preferably isopropyl
Alcohol.Usually portion isopropanol is equivalent to 2~2.5 parts of ethyl alcohol.It is especially suitable for that the situation of finite volume is added, DNA can be increased
Hydrophobic effect, promote DNA and silicon class medium microsphere joint efficiency.
It is further preferred that the chaotropic agent is selected from guanidine hydrochloride, guanidinium isothiocyanate, potassium iodide, potassium rhodanide, perchloric acid
Sodium, lithium chloride or combinations thereof are still more preferably guanidine hydrochloride or guanidinium isothiocyanate, are still more preferably guanidinium isothiocyanate.
Usual chaotrope concentration cannot be excessively high, will lead to protein denaturation and DNA degradation because excessively high, and be precipitated out from solution.It is high
The chaotropic agent of concentration can cause protein to open the secondary structures such as folding and increase protein and double-stranded DNA dissolution.Principle is logical
Often it is considered as the hydrogen bond in solution system where chaotropic agent destroys it, the protein of denaturation and DNA is promoted to show than it
The higher thermodynamic stability of native conformation, to guarantee that DNA is preferentially bound on silicon class medium microsphere.
Preferably, the silicon class medium microsphere suspension includes dispersion liquid and silicon class medium microsphere, and dispersion liquid is water, silicon class
Medium microsphere is silica bead, magnetic bead, diatom ooze or glass milk, further preferably silica bead.
Preferably, the microballoon of the silicon class medium microsphere suspension containing mass fraction 6%, 1~10 μm of partial size, microballoon warp
Acidizing pretreatment, pH value is less than 5, and further preferably 1.8.
It is further preferred that acidizing pretreatment is realized by the way that acid is added, the acid is inorganic acid or organic acid, inorganic acid choosing
From hydrochloric acid or nitric acid, organic acid is selected from acid or glacial acetic acid.
Still more preferably, acidizing pretreatment is realized by the way that hydrochloric acid is added, the specific steps are as follows:
1) 6g silicon class medium microsphere is weighed, 4 hours is stood after concussion dispersion in 100mL pure water, is repeated 2 times；
2) supernatant is removed, 1mol/L hydrochloric acid 50mL is added, stands 24 hours after concussion dispersion；
3) supernatant is removed, 100mL pure water is added as dispersion liquid, pH is adjusted to 1.8.Room temperature preservation.
Preferably, in the absorbability enhancer, the DNA short-movie section amplified production of external source addition is through such as SEQ ID NO.1
It expands and obtains with pair of primers shown in SEQ ID NO.2, fragment length is in the section 100-200bp, because of silicon class medium microsphere
It is lower to the adsorption efficiency of small fragment, to avoid the negative effect for having divided competitive binding microballoon with target dna.
5'-CAACAAAGCGAGTGTGGAGC-3', SEQ ID NO.1
5'-GTTCCTTGCGGGCATTTAGC-3', SEQ ID NO.2
Preferably, the proton in the absorbability enhancer or aprotic solvent are n,N-Dimethylformamide (DMF) or second
Nitrile, further preferred acetonitrile.
Preferably, the Rinse section includes rinsing liquid, is the ethanol water of volumetric concentration 60~90%.
Preferably, the multivalent anions in the efficient eluent are citrate ion, tartrate ion, phosphate radical
Any one of ion, nitrate ion, sulfate ion or salt acid ion are still more preferably phosphate anion.Cause
Also contain phosphate anion for DNA molecular, anion can usually play certain metalepsis, being both electronegative DNA
Get off from competitive elution on silicon class medium microsphere.
A method of ultramicron DNA extraction is carried out using mentioned reagent box, the specific steps are as follows:
(1) sample prepares: separately take a dry cotton swab to wipe again after taking the potential mottling in wet cotton swab lethal weapon surface,
Cotton swab head is fractureed and is transferred in centrifugation hand basket, hand basket is placed in mating centrifuge tube, and numbers record；
(2) cell cracking: lysate being added into sample, mixes, and metal dry bath or 56 DEG C of water-bath heat 30 minutes, upper machine
Hand basket is abandoned in centrifugation, leaves centrifuge tube and pyrolysis product therein in next step；
(3) DNA is adsorbed: combination buffer and silica bead suspension are added into centrifuge tube, absorbability enhancer is added after mixing,
Mix room temperature standing adsorption；
(4) it rinses desalination: being centrifuged after absorption, abandon supernatant to the greatest extent, rinsing liquid concussion is added and mixes, sufficiently removal salt
And impurity；
(5) elute: by rinsing liquid, centrifuge tube room temperature is dried after centrifugation is abandoned to the greatest extent, and volatilize remaining alcohol, and eluent, shake is added
It is eluted after swinging mixing, amplification or -20 DEG C of preservations is sucked out.
Preferably, when carrying out the DNA extraction in seminal stain, hair or nail, it should be added 0.03~0.05M's in lysate
Dithiothreitol (DTT), for destroying special cells film outer membrane structure.
Beneficial effects of the present invention:
Absorbability enhancer of the invention and efficient eluent are not only applicable to silica bead extracts kit, are equally applicable to be based on
Glassmilk, diatomite, magnetic bead DNA extraction kit.
It is improved based on silicon class medium microsphere ultramicron DNA extraction purification kit that the present invention relates to one kind, includes
Four cracking, absorption, rinsing, elution parts, cracking section are optimized mainly for except sample part, facilitate the same of operation
When obtain pyrolysis product to the maximum extent, design centrifuge tube and the matched hand basket with filtering function；Absorbed portion is main
A kind of absorbability enhancer has been invented, hydrophobic effect is increased, has eliminated pipe wall electrostatic effect, has promoted DNA and silicon class medium microsphere surface
Group effect, so that adsorption efficiency greatly improves；Elution fraction has then invented a kind of efficient eluent, innovatively using competition
Principle is eluted, DNA elution efficiency can be significantly improved, reduces the residual of silicon class medium microsphere surface DNA.The improved base of this case
Easy to operate in silicon class medium microsphere ultramicron DNA extraction purification kit, the DNA yield of extraction is high, and purity is high, segment are complete
Whole, in summary feature is especially suitable for the extraction of ultramicron biological material DNA, meets the high detection rate requirement increasingly proposed.
Reagent and kit provided by the present invention can not only be realized from various standard biologic sample (blood cake, seminal stain, salivas
Liquid spot, hair, tissue, cigarette butt, oral cavity cleaning piece, water bottle mouth cleaning piece) in extraction purification DNA, and can satisfy to trace amount DNA
The extraction requirement of (fingerprint, gloves print, mouse, cable, wallet, the cast-off cells left on the contacts sample such as mobile phone).It utilizes
The resulting DNA of reagent purification of the present invention can be applied to detect in next step without being further purified, such as
DNA is quantitative, be sequenced, STR is detected and SNP is detected etc..The DNA of reagent and its kits of the invention is especially suitable
It is detected together in PCR amplification.
Integrated operation of the present invention is simple, compares advantage with tradition silica bead method and embodies a concentrated reflection of cracking link, centrifuge tube and matches
The hand basket design function of set wonderfully simplifies step；High-volume is also not easy to make mistakes when extracting；Lytic effect is good, and guanidine hydrochloride substitutes different sulphur
Cyanic acid guanidine guarantees that the activity of Proteinase K makes the abundant cracking of cell to discharge the maximum amount of DNA；DNA mentions rate height, can reach
76% or more extraction efficiency.It is specific as follows:
1, the present invention is simplified cleavage step by a set of centrifuge tube and mating hand basket, and centrifugation directly abandons after cracking
DNA absorption link can be entered equipped with the sample after having cracked, operation simplifies, not easy to make mistakes.Hand basket of the invention has filtering
Function enables the pyrolysis product adhered on sample to drain completely under the influence of centrifugal force, and maximization avoids DNA from extracting
The direct losses of operating process are realized and farthest obtain DNA pyrolysis product, and DNA yield is improved.Lysate of the invention will
Guanidinium isothiocyanate has been replaced with guanidine hydrochloride, experiments verify that, guanidine hydrochloride is as a kind of denaturation more mild compared with guanidinium isothiocyanate
Agent, it is negligible to the activity influence of Proteinase K.
2, adsorption effect is good, and absorbability enhancer, including 3~10% protons or non-proton molten are increased on traditional formula
Agent, the DNA short-movie section amplified production of 80~120 μ g/mL external sources addition.When for ultramicron sample extraction nucleic acid, play efficiently
Adsorb the effect of nucleic acid.Because common test tube wall is all polyacrylic material, there is electrostatic on tube wall, nucleic acid can be adsorbed, mentioning
Taking in purification process can help to remove electrostatic effect using the DNA short-movie section amplified production of external source addition, enable DNA quilt very well
It adsorbs on silicon class medium microsphere, to reach recycling sample amplifying nucleic acid as big as possible；Proton or aprotic solvent can be into one
The hydrophobicity of step enhancing DNA promotes DNA, by disturbing, to increase absorption probability to silicon class medium microsphere.
3, elution efficiency is high, and negatively charged anion is added in the concept of innovative introduction of competition elution in eluent, can
Have and effectively same electronegative DNA is competed from silicon class medium microsphere, improves elution efficiency；Purification capacity is strong, energy
It is enough to be obtained in the sample for being mixed with a large amount of PCR inhibitors or pollutant sufficiently pure and downstream PCR, DNA survey be fully met requirements of
Sequence, the DNA of the operations such as real time fluorescent quantitative；Flexible reagent system, by expanding the operation system of reagent, it can be achieved that large volume
The enrichment of contained minim DNA sample in sample；The extensive biological sample scope of application, can be realized most of biological samples
DNA is extracted.
Detailed description of the invention
Fig. 1 is the trace occult blood spot STR parting map on lethal weapon；
Fig. 2 is mobile phone home key cast-off cells STR parting map.
Specific embodiment
The present invention will be further elaborated with reference to the accompanying drawings and examples, it should which explanation, following the description is only
It is not to be defined to its content to explain the present invention.
Embodiment 1:
The DNA and compliance test result in trace occult blood mottling on extraction purification lethal weapon
One, the DNA and STR of the potential blood mottling of trace on extraction purification lethal weapon are tested and analyzed
1, reagent is as follows:
Lysate: solvent is water, and solute includes following substance: the Tris-Hcl of 25mM, 1.5wt%triton x-100,
The Proteinase K of 10mMEDTA, 1.2M guanidine hydrochloride, the sodium chloride of 100mM and 2mg/ml, pH 7.5.Combination buffer: solvent is
Water, solute include following substance: the different sulphur cyanogen of the Tris-HCl of 100mM, 3wt%triton x-100,25mM EDTA, 5.7M
Sour guanidine, 30% isopropanol.PH 5.5, cooperation absorbability enhancer uses when use.
Absorbability enhancer: solvent is water, and solute includes following substance: 5% acetonitrile, the DNA of 110 μ g/ml external sources addition are short
Fragment amplification product.
Rinsing liquid: 75% ethanol water.
Eluent: solvent is water, and solute includes following substance: Tris-HCl, 1mM EDTA of 10mM, 2mM phosphoric acid hydrogen two
Sodium and sodium dihydrogen phosphate, pH 8.5.
Silicon class medium microsphere suspension: being the 6% processed silica bead of acid containing mass fraction, partial size 1-10um, and pH value is
1.8。
2, DNA is extracted with the reagent purification of step 1
Step 1) sample prepares: separately taking a dry cotton swab to wipe again after taking the potential mottling in wet cotton swab lethal weapon surface
It wipes, cotton swab head is fractureed and is transferred in centrifugation hand basket, hand basket is placed in mating centrifuge tube, and numbers record；
Step 2) cell cracking: the lysate of 300 μ L being added into sample, mixes, metal dry bath or 56 DEG C of water-bath heating
30 minutes, hand basket was abandoned in upper machine centrifugation, left centrifuge tube and pyrolysis product therein in next step；
Step 3) DNA absorption: 900 μ L combination buffers and 20-50 μ L silica bead suspension are added into centrifuge tube, after mixing
1 μ L absorbability enhancer is added, mixing room temperature standing adsorption, (note: absorbability enhancer must add after combination buffer in 10 minutes
Enter)；
Step 4) rinses desalination: 9000rcf abandons supernatant to the greatest extent after being centrifuged 1min after absorption, and the shake of 1mL rinsing liquid is added
Mixing is swung, salt and impurity are sufficiently removed；
If step 5) sample is dirtier, a rinse step can be added；
Step 6) elution: by rinsing liquid, centrifuge tube room temperature dries 8-10min after centrifugation is abandoned to the greatest extent, and volatilize remaining alcohol, root
According to needing to be added 20-200ul eluent, 56 DEG C of 1200rpm mix elution 10-15 minutes after concussion mixes, and amplification or -20 are sucked out
DEG C save.
3, STR is tested and analyzed
The DNA that step 2 is extracted carries out STR composite amplification, and composite amplification is using commercially available business STR composite amplification
Kit (the ID kit of such as American AB I company).Then it is detected with 3500 type genetic analyzers, as a result as shown in Figure 1, STR
Phenomena such as site is complete, and parting is accurate, no allelic loss, imbalance amplification, the peak Pull up, stutter band, the peak spike
Performance shows that the purification process purification efficiency is high, obtained DNA purity is high, and integrality is good.
Two, DNA adsorption efficiency is evaluated
It is used as initial DNA using 50,100,150,200ng DNA standard items (9947A, AGCU company), each concentration is set
Three repetitions.Adsorption efficiency measurement is carried out using the reagent that step 1 provides, Nanodrop 2000 measures the DNA in supernatant
Amount, and according to formula (adsorption efficiency=(amount of DNA (ng)/addition DNA standard items in amount-supernatant of DNA standard items are added
Amount (ng)) DNA adsorption efficiency is calculated.
The standard items DNA adsorption efficiency testing result of table 1, different additional amounts
Three, DNA extraction efficiency is evaluated
Using dose known amounts cell liquid as standard items, it is diluted to 200/μ L, takes 15 cotton swabs, three one group, altogether
It is divided into five groups, the cell dilution of 1 μ L, 2 μ L, 3 μ L, 4 μ L, 5 μ L is added dropwise in one to five groups respectively, after drying, provides using step 1
Reagent extracts efficiency test, and using identical elution volume, the DNA finally eluted is measured through Nanodrop 2000
As a result DNA content and OD260/280 value take every group of three duplicate average values.And according to formula (extraction efficiency=afford
Amount of DNA (ng)/addition DNA standard items amount (ng)) DNA extraction efficiency is calculated.DNA contains in the epithelial cell of people
Amount about 5.6 × 10-6μg。
The DNA concentration testing result extracted in table 2, different volumes cell diluent
Embodiment 2:
Extract the fingerprinting DNA and compliance test result on glass slide
1, clean 20 pieces of glass slide are chosen, choose any one finger pressing glass slide, dynamics is moderate and maintains 5-7s, often
Root finger corresponds to a glass slide, then adopts wet dry cotton swab method acquisition and carries the cast-off cells left on glass, using 1 step of embodiment
1 reagent provided carries out DNA extraction purification.
2, STR is tested and analyzed
The DNA that 2 step 1 of embodiment is extracted carries out STR composite amplification, and composite amplification is using commercially available business STR
Composite amplification reagent kit (the ID kit of such as American AB I company).Then it is detected with 3500 type genetic analyzers, as a result 20 fingers
Successfully detect that 6 pieces of complete maps, remaining sample have the phenomenon that locus loss in line sample, detection statistics the results are shown in Table
Shown in 3.
Table 3, simulation fingerprint sample STR testing result (STR bit is counted 16)
| Serial number | Detect STR number | Sample number |
| 1 | 16 | 6 |
| 2 | 14 | 8 |
| 3 | 10 | 3 |
| 4 | 8 | 3 |
Embodiment 3:
Extract cast-off cells DNA and compliance test result on mobile phone home key
1, it by after mobile phone home key ethanol for disinfection wiped clean, normal use 1-2 minutes, then adopts wet dry cotton swab method and adopts
The cast-off cells left on collection mobile phone home key carry out DNA extraction purification using the reagent that 1 step 1 of embodiment provides.
2, STR is tested and analyzed
The DNA that 3 step 1 of embodiment is extracted carries out STR composite amplification, and composite amplification is using commercially available business STR
Composite amplification reagent kit (the ID kit of such as American AB I company).Then it is detected with 3500 type genetic analyzers, STR bit point is complete
Whole, parting is accurate, without other problems in addition to slightly showing unbalanced, shows that purification process purifying DNA yield is high, integrality is good.Knot
Fruit is as shown in Figure 2.
Above-mentioned, although the foregoing specific embodiments of the present invention is described with reference to the accompanying drawings, not protects model to the present invention
The limitation enclosed, based on the technical solutions of the present invention, those skilled in the art are not needed to make the creative labor and can be done
Various modifications or changes out are still within protection scope of the present invention.
Sequence table
<110>Haining municipal public security bureau
Shanghai Jin Bo Bioisystech Co., Ltd
Zhongde Meilian Biotech Co., Ltd. Wuxi
<120>a kind of to be based on silicon class medium microsphere ultramicron DNA extraction purification kit
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
caacaaagcg agtgtggagc 20
<210> 2
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
gttccttgcg ggcatttagc 20
Claims (10)
1. one kind is based on silicon class medium microsphere ultramicron DNA extraction purification kit, including cracking section, absorbed portion, rinsing
Part and elution fraction, which is characterized in that the cracking section includes centrifuge tube and matched hand basket, is equipped in the bottom of hand basket
Filter membrane, the filter membrane stop moisture penetration by tension in the case where not being centrifuged；The absorbed portion includes combination buffer, silicon
Silicone hydroxyl, the solvent of absorbability enhancer are at least contained in class medium microsphere suspension, absorbability enhancer, the surface of silicon class medium microsphere
For water, solute includes: 3~10% proton of mass concentration or aprotic solvent, the DNA short-movie section of 80~120 μ g/mL external sources addition
Amplified production；The elution fraction includes efficient eluent, and the solvent of efficient eluent is water, and solute includes: 5~20mM's
Tris-HCl, 0.5~2mM chelating agent, 0.1~5mM multivalent anions, pH 8.0~9.0.
2. kit according to claim 1, which is characterized in that the cracking section further includes lysate, lysate
Solvent is water, and solute includes: the Tris-HCl of 5~50mM, 0.5~3wt% surfactant, 0.5~20mM chelating agent, 0.5
~1.5M guanidine hydrochloride, the sodium chloride of 50~120mM and the Proteinase K of 0.5~3mg/mL, pH 7.2~8.5.
3. kit according to claim 1, which is characterized in that the solvent of the combination buffer is water, and solute includes:
Tris-HCl, 1~5wt% surfactant, the chaotropic agent of 5~6M, 10~50mM chelating agent and the volumetric concentration of 40~150mM
20~35% alcohol content, pH 5.2~6.0.
4. kit according to claim 3, which is characterized in that the chaotropic agent is selected from guanidine hydrochloride, guanidinium isothiocyanate, iodine
Change potassium, potassium rhodanide, sodium perchlorate, lithium chloride or combinations thereof.
5. kit according to claim 1, which is characterized in that the silicon class medium microsphere suspension include dispersion liquid and
Silicon class medium microsphere, dispersion liquid are water, and silicon class medium microsphere is silica bead, magnetic bead, diatom ooze or glass milk.
6. kit according to claim 1, which is characterized in that the silicon class medium microsphere suspension contains mass fraction
6% microballoon, 1~10 μm of partial size, the acidified pretreatment of the microballoon, pH value is less than 5.
7. kit according to claim 1, which is characterized in that in the absorbability enhancer, the DNA short-movie of external source addition
Section amplified production is to expand to obtain through the pair of primers as shown in SEQ ID NO.1 and SEQ ID NO.2, and fragment length is in 100-
In the section 200bp.
8. kit according to claim 1, which is characterized in that proton or aprotic solvent in the absorbability enhancer
For n,N-Dimethylformamide or acetonitrile.
9. kit according to claim 1, which is characterized in that the multivalent anions in the efficient eluent are lemon
Any one of acid ion, tartrate ion, phosphate anion, nitrate ion, sulfate ion or salt acid ion.
10. a kind of method for carrying out ultramicron DNA extraction using kit described in any one of claim 1~9, feature exist
In, the specific steps are as follows:
(1) sample prepares: separately taking a dry cotton swab to wipe again after taking the potential mottling in wet cotton swab lethal weapon surface, by cotton
Label head, which fractures, to be transferred in centrifugation hand basket, and hand basket is placed in mating centrifuge tube, and numbers record；
(2) cell cracking: lysate being added into sample, mixes, and metal dry bath or 56 DEG C of water-bath heat 30 minutes, upper machine centrifugation
Hand basket is abandoned, leaves centrifuge tube and pyrolysis product therein in next step；
(3) DNA is adsorbed: combination buffer and silica bead suspension being added into centrifuge tube, absorbability enhancer is added after mixing, mixes
Room temperature standing adsorption；
(4) it rinses desalination: being centrifuged after absorption, abandon supernatant to the greatest extent, rinsing liquid concussion is added and mixes, sufficiently removes salt and miscellaneous
Matter；
(5) elute: by rinsing liquid, centrifuge tube room temperature is dried after centrifugation is abandoned to the greatest extent, and volatilize remaining alcohol, and eluent is added, and concussion is mixed
Amplification or -20 DEG C of preservations are sucked out in elution after even.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811019041.XA CN109280662A (en) | 2018-09-03 | 2018-09-03 | One kind being based on silicon class medium microsphere ultramicron DNA extraction purification kit |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811019041.XA CN109280662A (en) | 2018-09-03 | 2018-09-03 | One kind being based on silicon class medium microsphere ultramicron DNA extraction purification kit |
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| CN109280662A true CN109280662A (en) | 2019-01-29 |
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| CN201811019041.XA Pending CN109280662A (en) | 2018-09-03 | 2018-09-03 | One kind being based on silicon class medium microsphere ultramicron DNA extraction purification kit |
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