CN108949930A - VDR genetic polymorphism detection kit - Google Patents
VDR genetic polymorphism detection kit Download PDFInfo
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Abstract
The present invention provides a kind of VDR genetic polymorphism detection kit, comprising: MgCl2: 2.0~5.0mmol, dNTPs:0.2~0.8mmol, each primer: 0.1~1.0 μm of ol, each probe: 0.1~1.0 μm of ol, ABIFastmasterpremix:6~10 μ L, template: 10 μ L, total volume: 40 μ L.High sensitivity of the present invention can accurately detect the genomic DNA down to 1ng.Sample too long for the holding time and that these extracting concentrations of buccal swab are very low can be detected accurately, moreover, high specificity, is not in nonspecific result for the up to genomic DNA of 500ng or more.The process that diluted sample can be reduced, the directly upper mother liquor of the sample extracted can accurately be detected without non-specificity；Meanwhile being suitable for a variety of sample types: the present invention can not only detect the anticoagulant whole blood cells DNA of conventional EDTA, moreover it is possible to which the ropy buccal swab of Detection and Extraction, testing result accuracy are high.
Description
Technical field
The present invention relates to field of biotechnology, specially a kind of VDR genetic polymorphism detection kit.
Background technique
NP, single nucleotide polymorphism (full name SingleNucleotidePolymorphisms) refer to single in the genome
The genetic marker that the variation (including displacement, transversion, missing and insertion) of a nucleotide is formed, there are many quantity, rich polymorphism.
From the point of view of theoretically, each SNP site can have 4 kinds of different variant forms, but actually occur only there are two types of, i.e.,
Conversion and transversion, the ratio between the two are 1:2.SNP occurs the most frequently in CG sequence, and is mostly that C is converted to T, the reason is that CG
In C often be methylation, spontaneously become thymidine after deamination.In general, SNP refers to that variation frequency is greater than 1%
Single nucleotide variations.General every 1000 bases just have a SNP in human genome, and the SNP on human genome is total
Amount is probably 3 × 106.Therefore, SNP becomes third generation genetic marker, many phenotypic differences of human body, to the easy of drug or disease
Perception etc. all may be related with SNP.
Vitamin D receptor (VDR) is karyophilic protein, is mediation 1,25 (OH), it is big that D plays biology in the core of biological effect
Molecule belongs to superfamily member.Many biological functions of vitamin D are all to mediate to adjust target gene transcription come real by VDR
Existing 1,25 (OH), D hormone signal molecule form one receptor complex of hormone, compound effect in target cell in conjunction with VDR
In the specific dna sequence on target gene, adjustment effect is generated to the expression of structural gene.VDR be inherently a kind of ligand according to
Bad nuclear factor, it is maintaining one phosphorus metabolism of body calcium, adjusts cell Proliferation, differentiation etc. and plays an important role.
There are multiple endonuclease digestion sites in VDR gene order, it was confirmed that VDR gene has apparent polymorphism, arrives
So far, at least 25 VDR polymorphic sites are found, wherein study it is more for Bsm I, Apa I, Fok I this three
A site is the major site for participating in bone metabolism.The influence that the polymorphic site of different zones generates VDR gene expression is not
Together, Bsm I and Apa l restriction enzyme site are located on Section VIII introne, and polymorphism does not influence the amino acid sequence of VDR； Fok
I restriction enzyme site position transcription start site, polymorphism can lead to length amino acid sequence and change.
Summary of the invention
Technical problem solved by the invention is to provide a kind of VDR genetic polymorphism detection kit, above-mentioned to solve
The problems in background technique.
Technical problem solved by the invention is realized using following technical scheme: VDR genetic polymorphism detection kit,
It include: MgCl2: 2.0~5.0mmol, dNTPs:0.2~0.8mmol, each primer: 0.1~1.0 μm of ol, each probe: 0.1
~1.0 μm of ol, ABIFastmasterpremix:6~10 μ L, template: 10 μ L, total volume: 40 μ L.
VDR genetic polymorphism detection kit application method: the following steps are included:
Step (1), the DNA sample of extraction；
Step (2), DNA sample amplification: 2 parts of genomic DNAs, with TE diluted to concentration 1ng/ μ L, in ABI7300 fluorescence
It is expanded on quantitative PCR apparatus；Quantitative fluorescent PCR reaction system are as follows:
PCR reaction solution: MgCl2: 2.0~5.0mmol, dNTPs:0.2~0.8mmol, each primer: 0.1~1.0 μm of ol, respectively
Probe: 0.1~1.0 μm of ol, ABIFastmasterpremix:6~10 μ L, template: 10 μ L, total volume: 40 μ L.
PCR reaction condition are as follows: first stage: 95 DEG C of 5min；Second stage: 95 DEG C of 5s, 58 DEG C of 30s, 10 circulations；Third
Stage: 95 DEG C of 5s, 58 DEG C of 30s, 72 DEG C of 30s, 35 circulations；Phase III: when 35 circulations, FAM and JOE signal is collected；
Step (3), is as a result detected, detection method is as follows:
(1) every time in PCR reaction, while non-template control (NoTemplateControl is carried out；NTC), positive control and to be measured
The detection of sample；
(2) positive control is taken out to shake after thawing and is mixed, and be centrifuged 30s；
(3) 6 PCR reaction tubes are taken out and is centrifuged 30s after thawing, prevent reaction solution spillage when uncapping；
(4) ddH2O (NTC), sample to be tested, positive control are separately added into 6 PCR reaction tubes, every 10 μ L of hole；
(5) it the above 6 PCR reaction tube is covered into pipe is placed on centrifuge and be centrifuged 30S, it is ensured that be not smeared with drop on tube wall；
(6) 6 PCR reaction tubes are put in real-time PCR instrument；
PCR reaction condition are as follows: first stage: 95 DEG C of 4min；Second stage: 95 DEG C of 5s, 58 DEG C of 30s, 10 circulations；Third rank
Section: 95 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 30s, 35 circulations；Signal collection: FAM and JOE signal is collected at 72 DEG C of the phase III.
The DNA sample of the extraction is the extraction of clinical sample DNA, comprising the following steps:
1. handling blood-material:
A. when blood sample volume is less than 200 μ L, buffer GS can be added to supply volume to 200 μ L, then carry out next step experiment
(such as blood sample volume is 200 μ L, can directly carry out next step experiment, is not required to that GS is added)；B. when blood sample volume is more than
When 200 μ L, it need to be handled with cell pyrolysis liquid CL, the specific steps are as follows: the cell cracking of 1~2.5 times of volume is added in the sample
Liquid CL, is mixed by inversion, and 10,000rpm (~11,500 × g) are centrifuged 1min, suck supernatant, leaves nucleus precipitating (if cracking
It is not thorough, it is primary repeats above step), add 200 μ L buffer GS into the nucleus precipitating being collected by centrifugation, vibrates to thorough
Bottom mixes；
2. 20 μ LProteinaseK solution are added, mix；
3. adding 200 μ L buffer GB, sufficiently it is mixed by inversion, 56 DEG C of placement 10min are mixed by inversion for several times therebetween, and solution strain is clear
It is bright；
4. adding 200 μ L dehydrated alcohols, sufficiently it is mixed by inversion, at this time it is possible that flocculent deposit；
5. previous step acquired solution and flocculent deposit are all added in an adsorption column CB3 (adsorption column CB3 is put into collecting pipe),
12,000rpm (~13,400 × g) are centrifuged 30sec, outwell the waste liquid in collecting pipe, adsorption column CB3 is put into collecting pipe；
6. 500 μ L buffer GD (please first check whether before use and dehydrated alcohol has been added) are added into adsorption column CB3,12,
000rpm (~13,400 × g) is centrifuged 30sec, outwells the waste liquid in collecting pipe, adsorption column CB3 is put into collecting pipe；
7. 600 μ L rinsing liquid PW (please first check whether before use and dehydrated alcohol has been added) are added into adsorption column CB3,12,
000rpm (~13,400 × g) is centrifuged 30sec, outwells the waste liquid in collecting pipe, adsorption column CB3 is put into collecting pipe；
8. repetitive operation step 7；
9.12,000rpm (~13,400 × g) are centrifuged 2min, outwell waste liquid.Adsorption column CB3 is placed in and is placed at room temperature for several minutes,
Thoroughly to dry rinsing liquid remaining in adsorbent material；
10. adsorption column CB3 is transferred in 1.5mL centrifuge tube, 50-200 μ L elution buffer is vacantly added dropwise to adsorbed film middle position
Liquid TB, is placed at room temperature for 2~5min, and 12,000rpm (~13,400 × g) are centrifuged 2min, solution is collected into centrifuge tube；It is fixed
Amount: the DNA of extraction is quantified with ultraviolet specrophotometer, dilution DNA concentration to 1ng/ μ L.
The DNA sample of the extraction is buccal swab sample, comprising the following steps:
1. the cotton swab of buccal swab is cut from bar with scissors, it is placed in the centrifuge tube (providing for oneself) of 2mL, 400 μ is added
LBufferGR；If you need to the genomic DNA that no RNA pollutes, can be added RNaseA solution that 4 μ L concentration are 100mg/mL (article No.:
CW0601), concussion mixes；
2. 20 μ LProteinaseK and 400 μ LBufferGL are added, be vortexed concussion 15 seconds immediately, mixes well；It is added
It is mixed well immediately after BufferGL；ProteinaseK can not be directly added into BufferGL and be used；
3.56 DEG C are placed 10 minutes, and of short duration centrifugation makes the solution on tube wall be collected into tube bottom；
4. 400 μ L dehydrated alcohols are added, the concussion that is vortexed is mixed well, and of short duration centrifugation makes the solution on tube wall be collected into tube bottom；
5. upper step acquired solution and precipitating to be added in two portions to the adsorption column for having been charged into collecting pipe (CollectionTube2ml)
(SpinColumnDS) in, once it is no more than 700 μ L.12,000rpm (~13,400 × g) are centrifuged 1 minute, outwell collection
Waste liquid in pipe, adsorption column is placed back in collecting pipe；
6. 500 μ LBufferGW1 (whether preoperation inspection has been added dehydrated alcohol) is added into adsorption column, 12,000rpm from
The heart 1 minute, the waste liquid in collecting pipe is outwelled, adsorption column is placed back in collecting pipe；
7. 500 μ LBufferGW2 (whether preoperation inspection has been added dehydrated alcohol) is added into adsorption column, 12,000rpm from
The heart 3 minutes, the waste liquid in collecting pipe is outwelled, adsorption column is placed back in collecting pipe；It, can if you need to further increase DNA purity
Repeat this step；
8.12,000rpm centrifugations 1 minute, outwell the waste liquid in collecting pipe.Adsorption column is placed in several minutes of room temperature, thoroughly to dry in the air
It is dry；
9. adsorption column is placed in a new 1.5mL centrifuge tube, 50 μ are vacantly added to the intermediate position of adsorption column
LBufferGE or aqua sterilisa are placed at room temperature for 2~5 minutes, and DNA solution, -20 DEG C of preservations are collected in 12,000rpm centrifugations 1 minute；
10, quantitative, the DNA of extraction is quantified with ultraviolet specrophotometer, dilution DNA concentration to 1ng/ μ L.
It is compared to open technology, there are following advantages by the present invention:
(1) high sensitivity can accurately detect the genomic DNA down to 1ng.And buccal swab too long for the holding time this
A little very low samples of extracting concentration can be detected accurately.
(2) high specificity is not in nonspecific result for the up to genomic DNA of 500ng or more.It can be with
The process of diluted sample is reduced, the directly upper mother liquor of the sample extracted can accurately be detected without non-specificity.
(3) be suitable for a variety of sample types: the present invention can not only detect the anticoagulant whole blood cells DNA of conventional EDTA, also
The energy ropy buccal swab of Detection and Extraction, testing result accuracy are high.
(4) at low cost, ARMS typing can not only retain the high sensitivity of Taqman relative to Taqman probe typing
And high specific, and can also save the cost, the synthesis of ARMS primer is quick and easy, and synthesis cost is low, and expanding effect is more preferably.
(5) detection speed is fast, and entire detection process only needs 90 minutes.
(6) entire kit does not include poisonous and harmful substance, non-hazardous to operator and environment.
Detailed description of the invention
Fig. 1 is the high-resolution fusion curve figure of Bsm I site crt gene type of the invention；
Fig. 2 is the high-resolution fusion curve figure of Apa I site crt gene type of the invention；
Fig. 3 is the high-resolution fusion curve figure of Fok I site crt gene type of the invention.
Fig. 4 is the high-resolution fusion curve figure of Bsm I site crt gene type and sample of the invention 1. 2.
Fig. 5 is the high-resolution fusion curve figure of Apa I site crt gene type and sample of the invention 1. 2.
Fig. 6 is the high-resolution fusion curve figure of Fok I site crt gene type and sample of the invention 1. 2.
Fig. 7 is 1. 2. genotype that sample of the invention corresponds to three site Bsm I, Apa I, Fok I.
Specific embodiment
In order to make, technological means of the invention, creation characteristic, workflow, application method reach purpose and effect is easy to bright
White understanding, below in conjunction with the embodiment of the present invention, technical scheme in the embodiment of the invention is clearly and completely described,
In the description of the present invention, it should be noted that unless otherwise clearly defined and limited, term " installation ", " company ", " connection "
It shall be understood in a broad sense, for example, it may be being fixedly connected, may be a detachable connection, or be integrally connected to be mechanical connection,
It is also possible to be electrically connected;It can be directly connected, can also indirectly connected through an intermediary, it can company inside two elements
It is logical, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based on the present invention
In embodiment, every other implementation obtained by those of ordinary skill in the art without making creative efforts
Example, shall fall within the protection scope of the present invention.
Embodiment 1
The extraction of clinical sample DNA
One, EDTA anticoagulation
The present embodiment is genomic DNA to be extracted from EDTA anticoagulation, and quantify to it, the template as PCR detection.It adopts
With the poba gene group DNA extraction kit (TIANampDNABloodMiniKit) of Tiangeng (TIANGEN) company, according to reagent
Box illustrates to be operated, and specific details are as follows.
1. handling blood-material:
A. when blood sample volume is less than 200 μ L, buffer GS can be added to supply volume to 200 μ L, then carry out next step experiment
(such as blood sample volume is 200 μ L, can directly carry out next step experiment, is not required to that GS is added).
B. it when blood sample volume is more than 200 μ L, need to be handled with cell pyrolysis liquid CL, the specific steps are as follows: in sample
The middle cell pyrolysis liquid CL that 1~2.5 times of volume is added, is mixed by inversion, and 10,000rpm (~11,500 × g) are centrifuged 1min, sucks
Supernatant leaves nucleus precipitating (if cracking is not thorough, it is primary to repeat above step), heavy to the nucleus being collected by centrifugation
In shallow lake plus 200 μ L buffer GS, oscillation are mixed to thorough.
2. 20 μ LProteinaseK solution are added, mix.
3. adding 200 μ L buffer GB, sufficiently it is mixed by inversion, 56 DEG C of placement 10min are mixed by inversion for several times, solution is answered therebetween
Become limpid,
4. adding 200 μ L dehydrated alcohols, sufficiently it is mixed by inversion, at this time it is possible that flocculent deposit.
5. previous step acquired solution and flocculent deposit are all added in an adsorption column CB3, (adsorption column CB3 is put into collecting pipe
In), 12,000rpm (~13,400 × g) are centrifuged 30sec, outwell the waste liquid in collecting pipe, adsorption column CB3 is put into collecting pipe
In.
6. 500 μ L buffer GD (please first check whether before use and dehydrated alcohol has been added) are added into adsorption column CB3,
12,000rpm (~13,400 × g) are centrifuged 30sec, outwell the waste liquid in collecting pipe, adsorption column CB3 is put into collecting pipe.
7. 600 μ L rinsing liquid PW (please first check whether before use and dehydrated alcohol has been added) are added into adsorption column CB3,
12,000rpm (~13,400 × g) are centrifuged 30sec, outwell the waste liquid in collecting pipe, adsorption column CB3 is put into collecting pipe.
8. repetitive operation step 7.
9.12,000rpm (~13,400 × g) are centrifuged 2min, outwell waste liquid.Adsorption column CB3 is placed in and is placed at room temperature for several points
Clock, thoroughly to dry rinsing liquid remaining in adsorbent material.
10. adsorption column CB3 is transferred in 1.5mL centrifuge tube, 50-200 μ L elution is vacantly added dropwise to adsorbed film middle position
Buffer TB, is placed at room temperature for 2~5min, and 12,000rpm (~13,400 × g) are centrifuged 2min, solution is collected into centrifuge tube.
It is quantitative: the DNA of extraction to be quantified with ultraviolet specrophotometer, dilution DNA concentration to 1ng/ μ L.
Real-time fluorescence PCR method expands clinical sample DNA
The present embodiment is to use primer provided by the present invention, the extracted DNA sample of probe amplification embodiment 1.2 parts of genomes
DNA is expanded on ABI7300 fluorescence quantitative PCR instrument with TE diluted to concentration 1ng/ μ L with kit of the invention
Increase.
Quantitative fluorescent PCR reaction system are as follows:
PCR reaction solution:
MgCl2:2.0~5.0mmol
DNTPs:0.2~0.8mmol
Each primer: 0.1~1.0 μm of ol
Each probe: 0.1~1.0 μm of ol
The μ of ABIFastmasterpremix:6~10 L
Template: 10 μ L
Total volume: 40 μ L.
It is preferred that PCR reacts pipe fitting are as follows:
First stage: 95 DEG C of 5min；
Second stage: 95 DEG C of 5s, 58 DEG C of 30s, 10 circulations；
Phase III: 95 DEG C of 5s, 58 DEG C of 30s, 72 DEG C of 30s, 35 circulations；
Phase III: when 35 circulations, FAM and JOE signal is collected.
Detection method is as follows:
(1) every time in PCR reaction, while non-template control (NoTemplateControl is carried out；NTC), positive control and to be measured
The detection of sample.
(2) positive control is taken out to shake after thawing and is mixed, and be centrifuged 30s.
(3) 6 PCR reaction tubes are taken out and is centrifuged 30s after thawing, prevent reaction solution spillage when uncapping.
(4) ddH2O (NTC), sample to be tested, positive control are separately added into 6 PCR reaction tubes, every 10 μ L of hole.
(5) it the above 6 PCR reaction tube is covered into pipe is placed on centrifuge and be centrifuged 30S, it is ensured that be not smeared with drop on tube wall.
(6) 6 PCR reaction tubes are put in real-time PCR instrument.
The setting of reaction condition
First stage: 95 DEG C of 4min；
Second stage: 95 DEG C of 5s, 58 DEG C of 30s, 10 circulations；
Second stage: 95 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 30s, 35 circulations；
Signal collection: FAM and JOE signal is collected at 72 DEG C of the phase III.
Embodiment 2
Buccal swab sample
Genomic DNA is extracted from buccal swab, and it is quantified, the template as PCR detection.Use health for century
Buccal swab genome DNA extracting reagent kit, specific details are as follows.
1. the cotton swab of buccal swab is cut from bar with scissors, it is placed in the centrifuge tube (providing for oneself) of 2mL, 400 μ is added
LBufferGR。
Note: the RNaseA solution (goods that 4 μ L concentration are 100mg/mL can be added in the genomic DNA polluted if you need to no RNA
Number: CW0601), concussion mixes.
2. 20 μ LProteinaseK and 400 μ LBufferGL are added, be vortexed concussion 15 seconds immediately, mixes well.
Note: being mixed well immediately after BufferGL is added；ProteinaseK can not be directly added into BufferGL makes
With.
3.56 DEG C are placed 10 minutes, and of short duration centrifugation makes the solution on tube wall be collected into tube bottom.
4. 400 μ L dehydrated alcohols are added, the concussion that is vortexed is mixed well, and of short duration centrifugation makes the solution on tube wall be collected into pipe
Bottom.
5. upper step acquired solution and precipitating to be added in two portions to the suction for having been charged into collecting pipe (CollectionTube2ml)
In attached column (SpinColumnDS), once it is no more than 700 μ L.12,000rpm (~13,400 × g) are centrifuged 1 minute, are outwelled
Waste liquid in collecting pipe, adsorption column is placed back in collecting pipe.
6. 500 μ LBufferGW1 (whether preoperation inspection has been added dehydrated alcohol) is added into adsorption column, 12,
000rpm is centrifuged 1 minute, outwells the waste liquid in collecting pipe, adsorption column is placed back in collecting pipe.
7. 500 μ LBufferGW2 (whether preoperation inspection has been added dehydrated alcohol) is added into adsorption column, 12,
000rpm is centrifuged 3 minutes, outwells the waste liquid in collecting pipe, adsorption column is placed back in collecting pipe.
Note: if you need to further increase DNA purity, repeating step 7.
8.12,000rpm centrifugations 1 minute, outwell the waste liquid in collecting pipe.Adsorption column is placed in several minutes of room temperature, with thorough
It dries.
9. adsorption column is placed in a new 1.5mL centrifuge tube, 50 μ are vacantly added to the intermediate position of adsorption column
LBufferGE or aqua sterilisa are placed at room temperature for 2~5 minutes, and DNA solution, -20 DEG C of preservations are collected in 12,000rpm centrifugations 1 minute.
10, quantitative
The DNA of extraction is quantified with ultraviolet specrophotometer, dilution DNA concentration to 1ng/ μ L.
The present invention separately designs wild type and saltant type ARMS primer and Taqman-MGB probe for different genes site,
It is reacted in conjunction with quantitative fluorescent PCR, the genomic DNA extracted from human peripheral blood cell or buccal swab is detected, it can be with
It is disposably realized on a 6 PCR reaction item and gene pleiomorphism is detected, believed by being collected on real-time fluorescence PCR instrument
Number, the △ Ct value of wild type and saltant type is calculated to determine the genotype of sample DNA.
The above shows and describes the basic principle, main features and advantages of the invention.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this
The principle of invention, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, these changes
Change and improvement all fall within the protetion scope of the claimed invention.Claimed range of the invention by appended claims and
Its equivalent thereof.
Claims (4)
1. VDR genetic polymorphism detection kit, it is characterised in that: include: MgCl2: 2.0~5.0mmol, dNTPs:0.2~
0.8mmol, each primer: 0.1~1.0 μm of ol, each probe: 0.1~1.0 μm of ol, ABIFastmasterpremix:6~10
μ L, template: 10 μ L, total volume: 40 μ L.
2.VDR genetic polymorphism detection kit application method, it is characterised in that: the following steps are included:
Step (1), the DNA sample of extraction；
Step (2), DNA sample amplification: 2 parts of genomic DNAs, with TE diluted to concentration 1ng/ μ L, in ABI7300 fluorescence
It is expanded on quantitative PCR apparatus；Quantitative fluorescent PCR reaction system are as follows:
PCR reaction solution: MgCl2: 2.0~5.0mmol, dNTPs:0.2~0.8mmol, each primer: 0.1~1.0 μm of ol, each item
Probe: 0.1~1.0 μm of ol, ABIFastmasterpremix:6~10 μ L, template: 10 μ L, total volume: 40 μ L；
PCR reaction condition are as follows: first stage: 95 DEG C of 5min；Second stage: 95 DEG C of 5s, 58 DEG C of 30s, 10 circulations；Third rank
Section: 95 DEG C of 5s, 58 DEG C of 30s, 72 DEG C of 30s, 35 circulations；Phase III: when 35 circulations, FAM and JOE signal is collected；
Step (3), is as a result detected, detection method is as follows:
(1) every time in PCR reaction, while non-template control (NoTemplateControl is carried out；NTC), positive control and to be measured
The detection of sample；
(2) positive control is taken out to shake after thawing and is mixed, and be centrifuged 30s；
(3) 6 PCR reaction tubes are taken out and is centrifuged 30s after thawing, prevent reaction solution spillage when uncapping；
(4) ddH2O (NTC), sample to be tested, positive control are separately added into 6 PCR reaction tubes, every 10 μ L of hole；
(5) it the above 6 PCR reaction tube is covered into pipe is placed on centrifuge and be centrifuged 30S, it is ensured that be not smeared with drop on tube wall；
(6) 6 PCR reaction tubes are put in real-time PCR instrument.
3. VDR genetic polymorphism detection kit application method according to claim 2, it is characterised in that: the extraction
DNA sample be clinical sample DNA extraction, comprising the following steps:
1. handling blood-material:
A. when blood sample volume is less than 200 μ L, buffer GS can be added to supply volume to 200 μ L, then carry out next step experiment
(such as blood sample volume is 200 μ L, can directly carry out next step experiment, is not required to that GS is added)；B. when blood sample volume is more than
When 200 μ L, it need to be handled with cell pyrolysis liquid CL, the specific steps are as follows: the cell cracking of 1~2.5 times of volume is added in the sample
Liquid CL, is mixed by inversion, and 10,000rpm (~11,500 × g) are centrifuged 1min, suck supernatant, leaves nucleus precipitating (if cracking
It is not thorough, it is primary repeats above step), add 200 μ L buffer GS into the nucleus precipitating being collected by centrifugation, vibrates to thorough
Bottom mixes；
2. 20 μ LProteinaseK solution are added, mix；
3. adding 200 μ L buffer GB, sufficiently it is mixed by inversion, 56 DEG C of placement 10min are mixed by inversion for several times therebetween, and solution strain is clear
It is bright；
4. adding 200 μ L dehydrated alcohols, sufficiently it is mixed by inversion, at this time it is possible that flocculent deposit；
5. previous step acquired solution and flocculent deposit are all added in an adsorption column CB3 (adsorption column CB3 is put into collecting pipe),
12,000rpm (~13,400 × g) are centrifuged 30sec, outwell the waste liquid in collecting pipe, adsorption column CB3 is put into collecting pipe；
6. 500 μ L buffer GD (please first check whether before use and dehydrated alcohol has been added) are added into adsorption column CB3,12,
000rpm (~13,400 × g) is centrifuged 30sec, outwells the waste liquid in collecting pipe, adsorption column CB3 is put into collecting pipe；
7. 600 μ L rinsing liquid PW (please first check whether before use and dehydrated alcohol has been added) are added into adsorption column CB3,12,
000rpm (~13,400 × g) is centrifuged 30sec, outwells the waste liquid in collecting pipe, adsorption column CB3 is put into collecting pipe；
8. repetitive operation step 7；
9.12,000rpm (~13,400 × g) are centrifuged 2min, outwell waste liquid；Adsorption column CB3 is placed in and is placed at room temperature for several minutes,
Thoroughly to dry rinsing liquid remaining in adsorbent material；
10. adsorption column CB3 is transferred in 1.5mL centrifuge tube, 50-200 μ L elution buffer is vacantly added dropwise to adsorbed film middle position
Liquid TB, is placed at room temperature for 2~5min, and 12,000rpm (~13,400 × g) are centrifuged 2min, solution is collected into centrifuge tube；It is fixed
Amount: the DNA of extraction is quantified with ultraviolet specrophotometer, dilution DNA concentration to 1ng/ μ L.
4. VDR genetic polymorphism detection kit application method according to claim 2, it is characterised in that: the extraction
DNA sample be buccal swab sample, comprising the following steps:
1. the cotton swab of buccal swab is cut from bar with scissors, it is placed in the centrifuge tube (providing for oneself) of 2mL, 400 μ is added
LBufferGR；If you need to the genomic DNA that no RNA pollutes, can be added RNaseA solution that 4 μ L concentration are 100mg/mL (article No.:
CW0601), concussion mixes；
2. 20 μ LProteinaseK and 400 μ LBufferGL are added, be vortexed concussion 15 seconds immediately, mixes well；It is added
It is mixed well immediately after BufferGL；ProteinaseK can not be directly added into BufferGL and be used；
3.56 DEG C are placed 10 minutes, and of short duration centrifugation makes the solution on tube wall be collected into tube bottom；
4. 400 μ L dehydrated alcohols are added, the concussion that is vortexed is mixed well, and of short duration centrifugation makes the solution on tube wall be collected into tube bottom；
5. upper step acquired solution and precipitating to be added in two portions to the adsorption column for having been charged into collecting pipe (CollectionTube2ml)
(SpinColumnDS) in, once it is no more than 700 μ L；12,000rpm (~13,400 × g) are centrifuged 1 minute, outwell collection
Waste liquid in pipe, adsorption column is placed back in collecting pipe；
6. 500 μ LBufferGW1 (whether preoperation inspection has been added dehydrated alcohol) is added into adsorption column, 12,000rpm from
The heart 1 minute, the waste liquid in collecting pipe is outwelled, adsorption column is placed back in collecting pipe；
7. 500 μ LBufferGW2 (whether preoperation inspection has been added dehydrated alcohol) is added into adsorption column, 12,000rpm from
The heart 3 minutes, the waste liquid in collecting pipe is outwelled, adsorption column is placed back in collecting pipe；It, can if you need to further increase DNA purity
Repeat this step；
8.12,000rpm centrifugations 1 minute, outwell the waste liquid in collecting pipe；Adsorption column is placed in several minutes of room temperature, thoroughly to dry in the air
It is dry；
9. adsorption column is placed in a new 1.5mL centrifuge tube, 50 μ are vacantly added to the intermediate position of adsorption column
LBufferGE or aqua sterilisa are placed at room temperature for 2~5 minutes, and DNA solution, -20 DEG C of preservations are collected in 12,000rpm centrifugations 1 minute；
10, quantitative, the DNA of extraction is quantified with ultraviolet specrophotometer, dilution DNA concentration to 1ng/ μ L.
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Effective date of registration: 20190730 Address after: 226133 Biomedical Science and Technology Pioneer Park A9, Linjiang New District, Haimen City, Nantong City, Jiangsu Province Applicant after: Nantong Zhongke Gene Medical Testing Co., Ltd. Address before: 226000 Biomedical Science and Technology Pioneer Park A9, Linjiang Town, Haimen City, Nantong City, Jiangsu Province Applicant before: Shanghai GenomePilot Technology, Inc. |