CN101570778A - Method and kit for detecting polymorphism of ADPRT gene - Google Patents
Method and kit for detecting polymorphism of ADPRT gene Download PDFInfo
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- CN101570778A CN101570778A CNA2008100278825A CN200810027882A CN101570778A CN 101570778 A CN101570778 A CN 101570778A CN A2008100278825 A CNA2008100278825 A CN A2008100278825A CN 200810027882 A CN200810027882 A CN 200810027882A CN 101570778 A CN101570778 A CN 101570778A
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Abstract
The invention discloses a method for detecting the polymorphism of an ADPRT gene, which is characterized by comprising the following steps: (a) human genomic DNA is used as a template, and a positive primer and a reverse primer of a gene sequence are designed in a region of an ADPRT gene locus; a single basic group is mismatched on the positive primer so as to introduce an enzyme cutting site; and the template genomic DNA is processed by specificity polymerase chain reaction (PCR) amplification; (b) a restriction enzyme is utilized to carry out enzyme cutting on the genomic DNA; and (c) segment size separation is carried out on the ADPRT gene according to agarose gel electrophoresis so as to determine the gene polymorphism of the ADPRT gene. The invention also discloses a kit for detecting the ADPRT gene polymorphism, which comprises a gene polymorphism primer for amplification, dNTPs, DNA polymerase for the PCR reaction, a buffer solution of the DNA polymerase, a restriction enzyme Bsh1236I for restriction fragment length polymorphism (RFLP) and a corresponding buffer solution. The method has simple operation, low cost, intuitional judging result, quick reaction and convenient popularization and application, and the kit is convenient to use.
Description
Technical field
The present invention relates to a kind of gene tester, particularly a kind of test kit that detects detection method and this method of ADPRT gene pleiomorphism.
Background technology
The generation of tumour and differentiation are interactional multistage between environmental factors and the inherited genetic factors, the rapid mechanism of multistep.From macroscopical aspect, this process has embodied environment and host, especially with the interactional process of host's inherited genetic factors; And from the microcosmic aspect, the generation of tumour reflects the influence and the exercising result of carcinogenic factor pair cell genetic material in fact.Studies show that the someone suffers from cancer among the people who lives under equivalent environment, have the people safe and sound, even in some tumour hotspots, the actual sickness rate of tumour is also only about 0.1～0.2%.For many years, in the research of tumor aetiology, accumulated a large amount of data, illustrate that inherited genetic factors plays very important vital role in the generation of tumour, find that the earliest cancer may come from epidemiology by the evidence relevant with heredity, early stage investigation is found, in parent's generation or relatives born of the same parents, have among cancer patients's the people, the sickness rate of cancer does not have the people of this family history higher, and this further points out, and at least some tumours are directly related with heredity.
Under the same contact conditions, the individual susceptibility of tumour is different.The tumor susceptibility influence factor mainly is that metabolic enzyme differential expression, DNA repair ability difference and proto-oncogene or expression of tumor suppressor gene are unusual.Tumour be that environmental factors acts on the result that genetic material such as DNA cause dna damage, so that injury repairing, particularly dna damage are repaired is significant.And individual DNA repair ability and its DNA-repair gene have substantial connection, and the crowd DNA-repair gene of studies show that has different polymorphisms, can show as different inherited character.
Gene pleiomorphism be meant as base replace, excision/insertion and mutant dna sequence that gene replication/excision etc. causes, they can cause protein function and phenotypic change.Most gene pleiomorphisms are positioned at non-coding region, thereby not significantly effect; If but gene pleiomorphism occurs in the inside of gene coding region-exon, amino acid whose replacement just may take place in this site so and cause protein active change in various degree; The polymorphism of promoter region can change the speed of transcribing, and the polymorphism that is positioned at intron and exon border can cause to transcribe and produce wrong mRNA segment, thereby not exclusively synthetic or do not have active albumen.The polymorphism that is characteristics with whole fragment gene excision can cause the active forfeiture of functional enzyme, as the blank allelotrope of glutathione-S-transferase M1 (GSTM1); The polymorphism that whole fragment gene duplicates then makes proteins encoded produce higher levels of enzymic activity.The increase that single nucleotide polymorphism (SNPs) can cause synonym or non-synonym amino acid to be replaced.Synonym replace be meant an amino acid by a difference but similar amino acid replace because similar amino acid is by relevant codon coding, activity of proteins is difficult for changing; So the change meeting of synonym base at random produces minimum mutation effect to protein, and is also less to the harm of human body; But not synonym SNPs (the amino acid whose replacements that chemical property is different) may produce deleterious effect to human body.
ADPRT is then bringing into play direct and important effect in the dna damage repair process.The ADPRT gene is positioned at No. 1 karyomit(e) of people, total length 47286bp, have 23 exons and 22 introns, wherein 762 codons of the 17th exon are the more common gene polymorphic sites relevant with tumor susceptibility, the SNP site of ADPRT the 762nd bit codon T → C is non-synonym SNP, can cause that the 762nd amino acids becomes Ala by Val, may influence the proteic function of the ADPRT with important repairing effect.Studies show that ADPRT Ala762Ala genotype can make Aisa people and Caucasia crowd's ADP ribosyltransferase vigor descend 40%, it is relevant with the dangerous increase of tumour that the ADPRT enzyme activity reduces, and the polymorphic generation with squamous cell carcinoma of esophagus, stomach carcinoma of gastric cardia and the relevant lung cancer of smoking of Chinese population ADPRT Val762Ala is relevant.Carry the genotypic Caucasian of ADPRT Ala762Ala and suffer from risk of prostate cancer and obviously increase, and its risk level is relevant with the number of carrying mutator gene.
In recent years, people have been developed many methods that are used to detect gene pleiomorphism, more common have single-strand conformation polymorphism technology (SSCP), directly sequencing technologies, sex change one high pressure liquid chromatography (DHPLC), dynamically equipotential gene specific hybridization technology (DASH) and a TagMan technology etc., wherein a lot of methods are carrying out aspect the high throughput testing very remarkable advantages being arranged, but owing to instrument costliness, requirement have the support of corresponding techniques platform, it is relatively also higher to detect cost, and is not suitable for the sudden change detection of small sample known site.
Summary of the invention
The objective of the invention is in order to solve weak point of the prior art, a kind of ADPRT gene pleiomorphism that is used to detect is provided, simple to operate, specificity is high, good reproducibility, the position and the character of sudden change can be directly determined, detection method and the test kit of a large amount of samples, funds and time can be saved.
In order to achieve the above object, the present invention adopts following scheme:
A kind of detection method that detects the ADPRT gene pleiomorphism is characterized in that this detection method may further comprise the steps:
A, be template, at the forward and reverse primer of ADPRT gene locus region design gene order, introduce a restriction enzyme site at forward primer, and templet gene group DNA is carried out the specific PCR amplification by single base mismatch with human gene group DNA;
B, utilize restriction enzyme that it is carried out enzyme to cut;
C, according to agarose gel electrophoresis above-mentioned ADPRT gene is carried out clip size and separate, determine its gene pleiomorphism;
Wherein said ADPRT gene locus is Val762Ala (rs1136410), and described forward primer is: 5 '-TTTTGCTCCTCCAGGCCAAC*G-3 ' (base before the * for the base mismatch introduced to create restriction enzyme site); Described reverse primer is: 5 '-CCTGACCCTGTTACCTTAATGTCAGTTTT-3 '.
Aforesaid detection method, wherein the pcr amplification described in the step a is under 25 μ l reaction systems, behind 94 ℃ of pre-sex change 5min, carries out 30 following cyclic amplifications: 94 ℃ of sex change 40s, 57 ℃ of annealing 40s, 72 ℃ are extended 35s, and last 72 ℃ are extended 5min.
Aforesaid detection method, wherein said 25 μ l reaction systems comprise: 1U TaqDNA polysaccharase, 10 * Buffer, 2.5 μ l, 25mol/L MgCl
22 μ l, 2.5mmol/LdNTPs 2.5 μ l, the 50ng human gene group DNA, each 1 μ l of 10 μ mol/L upstream and downstream primers supplies 25 μ l by the sterilization ultrapure water.
Aforesaid detection method, wherein the restriction enzyme described in the step b is that 20 μ l Bsh1236I enzymes are cut system, the described 20 μ l Bsh1236I enzyme systems of cutting comprise: the pcr amplification product of 10 μ l, the Bsh1236I restriction enzyme 1 μ l of 10U/ μ l, 10 * damping fluid, 2 μ l and 7 μ l sterilization ultrapure water.
A kind of test kit that detects the ADPRT gene pleiomorphism, this test kit comprise the usefulness that increases gene-specific primer, dNTPs, be used for the archaeal dna polymerase and the damping fluid thereof of PCR reaction, be used for restriction enzyme Bsh1236I and the corresponding damping fluid of RFLP.
Aforesaid test kit, this test kit comprises:
Forward primer is: 5 '-TTTTGCTCCTCCAGGCCAACG-3 ';
Reverse primer is: 5 '-CCTGACCCTGTTACCTTAATGTCAGTTTT-3 ';
1U Taq archaeal dna polymerase;
10×Buffer?2.5μl；
25mol/L?MgCl
2?2μl；
2.5mmol/L?dNTPs?2.5μl；
Each 1 μ l of 10 μ mol/L upstream and downstream primers.
10 Bsh1236I of unit enzymes;
The enzyme cutting buffering liquid of 2 μ L.
In sum, beneficial effect of the present invention:
Detection method of the present invention belongs to polymerase chain reaction-restriction fragment length polymorphism (polymerase chain reaction-restriction fragment lengthpolymorphism, PCR-RFLP) detection technique, it has simple to operate, advantages such as specificity is high, good reproducibility, and can directly determine the position and the character of sudden change to be specially adapted to the detection in some small sample known mutations sites.Detection method of the present invention is introduced base mismatch and is created restriction enzyme site (create restriction site, CRS) method designs suitable primer, carry out PCR-RFLP then and detect the too expensive problem that has solved the restriction endonuclease price, this detection method has very big handiness in actual applications, some advantages that kept simultaneously the RFLP method again well, has application promise in clinical practice, originally the no-go project that can't detect or cost an arm and a leg can be realized, and can save a large amount of samples, funds and time, correlative study is produced good promoter action.
Embodiment
Below in conjunction with embodiment the present invention is further described:
A kind of detection method that detects the ADPRT gene pleiomorphism of the present invention, this detection method may further comprise the steps:
A, be template, at the forward and reverse primer of ADPRT gene locus region design gene order, introduce a restriction enzyme site at forward primer, and templet gene group DNA is carried out the specific PCR amplification by single base mismatch with human gene group DNA;
B, utilize restriction enzyme that it is carried out enzyme to cut;
C, according to agarose gel electrophoresis above-mentioned ADPRT gene is carried out clip size and separate, determine its gene pleiomorphism;
Wherein said ADPRT gene locus is Val762Ala (rs1136410), and described forward primer is: 5 '-TTTTGCTCCTCCAGGCCAAC*G-3 ' (base before the * for the base mismatch introduced to create restriction enzyme site); Described reverse primer is: 5 '-CCTGACCCTGTTACCTTAATGTCAGTTTT-3 '.
Carry out above-mentioned detection among the present invention and use following test kit, this test kit comprise the usefulness that increases gene-specific primer, dNTPs, be used for the archaeal dna polymerase and the damping fluid thereof of PCR reaction, be used for restriction enzyme Bsh1236I and the corresponding damping fluid of RFLP.Specifically comprise:
Forward primer is: 5 '-TTTTGCTCCTCCAGGCCAACG-3 ';
Reverse primer is: 5 '-CCTGACCCTGTTACCTTAATGTCAGTTTT-3 ';
1U Taq archaeal dna polymerase;
10×Buffer?2.5μl；
25mol/L?MgCl
2?2μl；
2.5mmol/L?dNTPs?2.5μl；
Each 1 μ l of 10 μ mol/L upstream and downstream primers.
10 Bsh1236I of unit enzymes;
The enzyme cutting buffering liquid of 2 μ L.
The genomic dna that detection method of the present invention is used is extracted by following method:
The solution preparation
1. Solution I:38mmol/LNaCl, 10mmol/LEDTA, 5mmol/LTris-HCl (pH8.0), autoclaving, room temperature preservation.
2. Solution II:150mmol/LNaCl, 10mmol/LEDTA, 5mmol/LTris-HCl (pH8.0), 1%Triton-X-100,2%SDS, autoclaving, room temperature preservation.
3. TE damping fluid: 10mmol/L Tris-HCl (pH7.5), 1mmol/L EDTA (pH8.0).Autoclaving, 4 ℃ of preservations.
4. Proteinase K: be made into 20mg/ml solution with distilled water, packing is standby ,-20 ℃ of preservations.
5. chloroform: primary isoamyl alcohol (24: 1): the volume ratio with 24: 1 is mixed with mixed solution, is kept in the brown bottle room temperature preservation.
6. 3mmol/L NaAc (pH4.8-5.0): 61.5g NaAc is dissolved in the 120ml distilled water, is settled to 250ml, regulates the pH value to 4.8-5.0 with concentrated hydrochloric acid then.
7. 75% ethanol: 75ml dehydrated alcohol water is settled to 100ml, preserves in-20 ℃ of refrigerators.
8. ethidium bromide: be made into the 10mg/ μ l aqueous solution, lucifuge is preserved in 4 ℃ of refrigerators.
9. 6 * sample-loading buffer: be made into the aqueous solution that contains 0.25% tetrabromophenol sulfonphthalein and 30% glycerine, preserve in 4 ℃ of refrigerators.
10. 5 * tbe buffer liquid: Tis54g, boric acid 27.5g, 0.5mol/L EDTA 20m mixes, and distilled water is settled to 1L, room temperature preservation.During electrophoresis, with 10 times of distilled water dilutings.
Working method:
1. blood clot is transferred in the tissue homogenizer, adds a small amount of Solution I, fully grind, blood clot is made homogenate;
2. homogenate is transferred in the 50ml polypropylene centrifuge tube, adds Solution I to 30ml, put upside down centrifuge tube for several times, make homogenate and mixed solution thorough mixing.Ice bath 5-10min;
3. the centrifugal 15min of 3000rpm outwells supernatant liquor gently;
4. repeating step 2. and 3., till precipitation does not take on a red color;
5. after abandoning supernatant, add 2.5ml Solution II, 25 μ l Proteinase K solution and 160 μ l 10%SDS in the 50ml centrifuge tube, shake up gently, 37 ℃ of water-baths are spent the night;
6. the mixture after digestion being spent the night is transferred in the 10ml centrifuge tube;
7. add isopyknic balance phenol in the Digestive system, cover tight lid, shake 5min gently, make the thorough mixing of phenol and water, the centrifugal 5min of 3000rpm carefully draws the upper strata water to another 10ml centrifuge tube with dropper.As above repeat once with the balance phenol extracting.
8. the upper strata aqueous phase adds isopyknic chloroform: primary isoamyl alcohol (24: 1), 3000rpm, 4 ℃ of centrifugal 5min.As above repeat to use chloroform and primary isoamyl alcohol extracting once.
9. upper water is moved into mutually in the 10ml tool plug glass centrifuge tube, and recording volume, add the 3mmol/L NaAc (pH4.8-5.0) of 1/10 volume, jiggle and make it mixing.Add the cold dehydrated alcohol (20 ℃) of 2.5 times of volumes then, shake centrifuge tube gently, visible white cotton-shaped chromosomal DNA is separated out.
10. use the heavy caliber dropper with the DNA sucking-off, put into the 1.5ml centrifuge tube, cold ethanol with 75% washs DNA gently, the centrifugal 5min of 1000rpm discards ethanolic soln, places under the room temperature about 2h, make the DNA drying, add an amount of TE damping fluid (150 μ l) then, place a couple of days, DNA is dissolved fully for 4 ℃.
5 times of DNA dilutions after will dissolve, on 1.0% sepharose, electrophoresis 1h under 100V, the 50mA condition takes pictures under gel systems then.
After DNA concentration and purity testing: DNA dissolves fully, get 5 μ lDNA solution, add 95 μ lTE damping fluids, fully mixing.Adopt the nucleic acid-protein tester to measure,,, calculate DNA concentration and purity in wavelength 260nm and 280nm place difference photometry density with the TE zeroing.The OD260/280 ratio of DNA should be about 1.80.As＜1.80, illustrate wherein and may contain protein; As＞1.80, illustrate wherein and may contain RNA.When not only having contained protein but also having contained RNA in the solution, this ratio may just in time be 1.80 also.Wavelength 260nm place, 1OD are equivalent to 50 μ g/ml double-stranded DNAs, so DNA concentration=OD260 * 50 * extension rate (unit: μ g/ml).
Embodiment 1
Genomic dna with said extracted is a template, at the forward and reverse primer of ADPRT gene locus Val762Ala (rs1136410) region design gene order, described forward primer is: 5 '-TTTTGCTCCTCCAGGCCAAC*G-3 ' (base before the * for the base mismatch introduced to create restriction enzyme site); Described reverse primer is: 5 '-CCTGACCCTGTTACCTTAATGTCAGTTTT-3 ', introduce a restriction enzyme site by single base mismatch at forward primer, and templet gene group DNA carried out the specific PCR amplification; Concrete pcr amplification step is as follows:
To sample gene group DNA, carry out pcr amplification with above-mentioned positive and negative primer according to following reaction system and condition: 25 μ l reaction systems, comprise 1U Taq archaeal dna polymerase, 10 * Buffer2.5 μ l, 25mol/L MgCl22 μ l, 2.5mmol/L dNTPs 2.5 μ l, 50ng human genome DNA, each 1 μ l of 10 μ mol/L upstream and downstream primers supplies 25 μ l by the sterilization ultrapure water.Behind 94 ℃ of pre-sex change 5min, carry out 30 and carry out 30 following cyclic amplifications: 94 ℃ of sex change 40s, 57 ℃ of annealing 40s, 72 ℃ are extended 35s, and last 72 ℃ are extended 5min.DNA sample to above-mentioned amplification is cut carrying out enzyme in the 20 μ l Bsh1236I enzymes system of cutting, the wherein said 20 μ l Bsh1236I enzyme systems of cutting comprise: the pcr amplification product of 10 μ l, the Bsh1236I restriction enzyme 1 μ l of 10U/ μ l, 10 * damping fluid, 2 μ l.
Get PCR product 10 μ L, add the enzyme cutting buffering liquid of 10 Bsh1236I of unit enzymes and 2 μ L, form 20 μ L reaction systems, after 37 ℃ of water-bath digested overnight at 2% agarose gel electrophoresis 30min, the evaluation product of under ultraviolet lamp, taking pictures.T/T genotype person does not have the Bsh1236I restriction enzyme site, shows 157bp one band, and C/C genotype person has restriction enzyme site, and 136bp and 21bp two bands are arranged.And the T/C heterozygote shows above-mentioned 157bp, 136bp and 21bp band.
Claims (6)
1, a kind of detection method that detects the ADPRT gene pleiomorphism is characterized in that this detection method may further comprise the steps:
A, be template, at the forward and reverse primer of ADPRT gene locus region design gene order, introduce a restriction enzyme site at forward primer, and templet gene group DNA is carried out the specific PCR amplification by single base mismatch with human gene group DNA;
B, utilize restriction enzyme that it is carried out enzyme to cut;
C, according to agarose gel electrophoresis above-mentioned ADPRT gene is carried out clip size and separate, determine its gene pleiomorphism;
Wherein said ADPRT gene locus is Val762A1a (rs1136410), and described forward primer is: 5 '-TTTTGCTCCTCCAGGCCAAC*G-3 ' (base before the * for the base mismatch introduced to create restriction enzyme site); Described reverse primer is: 5 '-CCTGACCCTGTTACCTTAATGTCAGTTTT-3 '.
2, detection method according to claim 1, wherein the pcr amplification described in the step a is under 25 μ l reaction systems, behind 94 ℃ of pre-sex change 5min, carry out 30 following cyclic amplifications: 94 ℃ of sex change 40s, 57 ℃ of annealing 40s, 72 ℃ are extended 35s, and last 72 ℃ are extended 5min.
3, detection method according to claim 2, wherein said 25 μ l reaction systems comprise: 1U Taq archaeal dna polymerase, 10 * Buffer, 2.5 μ l, 25mol/L MgCl
22 μ l, 2.5mmol/L dNTPs 2.5 μ l, the 50ng human gene group DNA, each 1 μ l of 10 μ mol/L upstream and downstream primers supplies 25 μ l by the sterilization ultrapure water.
4, detection method according to claim 1, wherein the restriction enzyme described in the step b is that 20 μ l Bsh1236I enzymes are cut system, the described 20 μ l Bsh1236I enzyme systems of cutting comprise: the pcr amplification product of 10 μ l, the Bsh1236I restriction enzyme 1 μ l of 10U/ μ l, 10 * damping fluid, 2 μ l and 7 μ l sterilization ultrapure water.
5, a kind of test kit that detects the ADPRT gene pleiomorphism, this test kit comprise the usefulness that increases gene-specific primer, dNTPs, be used for the archaeal dna polymerase and the damping fluid thereof of PCR reaction, be used for restriction enzyme Bsh1236I and the corresponding damping fluid of RFLP.
6, test kit according to claim 5, this test kit comprises:
Forward primer is: 5 '-TTTTGCTCCTCCAGGCCAAC*G-3 ';
Reverse primer is: 5 '-CCTGACCCTGTTACCTTAATGTCAGTTTT-3 ';
1U Taq archaeal dna polymerase;
10×Buffer?2.5μl；
25mol/L?MgCl
22μl；
2.5mmol/L?dNTPs?2.5μl；
Each 1 μ l of 10 μ mol/L upstream and downstream primers.
10 Bsh1236I of unit enzymes;
The enzyme cutting buffering liquid of 2 μ L.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102787160A (en) * | 2011-05-17 | 2012-11-21 | 中国科学院上海生命科学研究院 | Method for high throughput detection of gene polymorphism |
| CN105087818A (en) * | 2015-09-24 | 2015-11-25 | 郑州大学 | Method and kit for determining POT1 gene rs1034794 locus polymorphism |
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| CN105274203A (en) * | 2015-09-24 | 2016-01-27 | 河南省职业病防治研究院 | Method and kit for determination of human NEFL gene rs1059111 site polymorphism |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102787160A (en) * | 2011-05-17 | 2012-11-21 | 中国科学院上海生命科学研究院 | Method for high throughput detection of gene polymorphism |
| CN105087818A (en) * | 2015-09-24 | 2015-11-25 | 郑州大学 | Method and kit for determining POT1 gene rs1034794 locus polymorphism |
| CN105274211A (en) * | 2015-09-24 | 2016-01-27 | 河南省职业病防治研究院 | Method and kit for determination of human NEFM gene polymorphism rs12515 site polymorphism |
| CN105274216A (en) * | 2015-09-24 | 2016-01-27 | 河南省职业病防治研究院 | Method and kit for determining rs2979704 site polymorphism of human NEFL gene |
| CN105274203A (en) * | 2015-09-24 | 2016-01-27 | 河南省职业病防治研究院 | Method and kit for determination of human NEFL gene rs1059111 site polymorphism |
| CN107119133A (en) * | 2017-05-22 | 2017-09-01 | 安徽大学 | Non-diseases diagnoses the method and kit with the detection leptin gene polymorphism of therapeutical uses |
| CN110438211A (en) * | 2019-08-20 | 2019-11-12 | 福建晨欣科生物科技有限公司 | Specific gene segment enrichment detection kit and method based on digestion PCR |
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