CN104878119A - Human ALDH 2 gene detection kit - Google Patents
Human ALDH 2 gene detection kit Download PDFInfo
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- CN104878119A CN104878119A CN201510359801.1A CN201510359801A CN104878119A CN 104878119 A CN104878119 A CN 104878119A CN 201510359801 A CN201510359801 A CN 201510359801A CN 104878119 A CN104878119 A CN 104878119A
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Abstract
The invention relates to the field of biotechnologies, in particular to a human ALDH2 gene detection kit. The human ALDH2 gene detection kit comprises a wild type ALDH2 gene specific amplification primer pair, a mutant type ALDH2 gene specific amplification primer pair and TspRI restriction enzyme. By the adoption of the detection kit, no extra sequenator or QPCR instrument or chip scanning instrument needs to be purchased, and detection can be conducted in amounts of common molecule labs. Meanwhile, compared with other detection methods, the human ALDH2 gene detection kit further has the advantages of being quick, cheap, stable, precise and comprehensive.
Description
Technical field
The present invention relates to biological technical field, be specifically related to a kind of mankind ALDH2 gene detecting kit.
Background technology
Aldehyde dehydrogenase 2 (ALDH2) plays an important role in the alcohol metabolism process of human body.After people takes in alcohol, first the ethanol dehydrogenase in body is oxidized to acetaldehyde, then by ALDH2, acetaldehyde is transformed into acetic acid further.Acetaldehyde has stronger toxicity to health, once acetaldehyde dehydrogenase is undergone mutation, will affect the efficiency of health degrade acetaldehyde, acetaldehyde is caused to be accumulated in vivo, thus cause uncomfortable, if things go on like this will cause liver, nephrocardiac burden, thus cause various diseases.There are some researches show, the sudden change of aldehyde dehydrogenase 2 and the in close relations of all kinds of alimentary tract cancer.Simultaneously, the key of aldehyde dehydrogenase 2 or effectively metabolism pannonit, and the crowd of aldehyde dehydrogenase 2 sudden change takes pannonit Risk of noneffective and significantly increases, so need to carry out aldehyde dehydrogenase 2 gene test before taking pannonit, if it is invalid that detected result shows that person under inspection takes pannonit, then need to use other medicines instead.
In prior art, the existing technology detecting ALDH2 transgenation.Such as: (1) adopts the method for gene chip to carry out ALDH2 detection in Gene Mutation, this technology at the base difference Position Design oligonucleotide sequence of wild-type and saltant type, by whether differentiating that detected object is wild-type or saltant type with chip hybridization.Technological step based on gene chip is loaded down with trivial details, and the treatment time of each sample is longer, is difficult to the batch processing of carrying out large sample amount, and the cost that single detects is higher, also needs chip scanner to diagnose simultaneously, needs to purchase extra test set.(2) adopt the method for generation order-checking to distinguish wild-type and saltant type, described technology needs generation sequenator to check order, and experimental period is longer simultaneously.(3) adopt the method for QPCR to distinguish wild-type and saltant type, but described technology need QPCR instrument to check order, complicated operation.(4) carry out PCR with specific primer and distinguish wild-type and saltant type, but the accuracy rate of described technology is low.
Summary of the invention
For defect existing in prior art, the object of the invention is to, provide a kind of and detect the ALDH2 gene detecting kit quick, accuracy rate is high, simple to operate, cost is low.
Another object of the present invention is to, institute's detection kit application and using method thereof are provided.
The present invention is achieved by the following technical solutions:
A first aspect of the present invention, provides a kind of ALDH2 gene detecting kit, comprises wild-type and saltant type ALDH2 gene-specific amplification primer pair and TspRI restriction enzyme.
Preferably, described primer pair comprises forward primer and reverse primer, and the nucleotide sequence of forward primer, as shown in SEQ ID NO.3, is specially: 5 '-TGTAACCCATAACCCCCAAG-3 '; The nucleotide sequence of reverse primer, as shown in SEQ ID NO.4, is specially: 5 '-AAACACTGATGGCCTCAAGC-3 '.
The key of test kit of the present invention is combinationally using first of described primer pair and described restriction enzyme.The present inventor, through repeatedly comparing and optimizing, obtains the primer pair of described can simultaneously increase wild-type and saltant type ALDH2 gene.According to single base difference site of wild-type and saltant type ALDH2 gene, devise described TspRI restriction enzyme, wild-type ALDH2 gene can be digested by described restriction enzyme identification, and restriction enzyme described in saltant type ALDH2 gene pairs is insensitive, therefore digestion can not be identified.Postdigestive product is directly carried out electrophoresis, if electrophoresis result only has the band of complete an about 400bp, then illustrates that two copies of the ALDH2 gene of experimenter can not, by TspRI digestion with restriction enzyme, be all homozygous mutant ALDH2 gene; If electrophoresis result has two digested bands, then two copies of the ALDH2 gene of experimenter can, by TspRI digestion with restriction enzyme, be all wild-type ALDH2 gene; If electrophoresis result has three bands (wherein having the not digested band of an about 400bp and two digested bands), then the ALDH2 gene of experimenter has a copy can by TspRI digestion with restriction enzyme, one can not, by TspRI digestion with restriction enzyme, be heterozygous mutant ALDH2 gene.
Based on described test kit employing of the present invention is that PCR carries out detection by quantitative, some other reagent can also be comprised in described test kit, as: one or more in STb gene extraction agent, PCR reagent, PCR primer purified reagent, gel electrophoresis reagent and reference substance.Specifically need which reagent to be fitted into test kit, can configure according to actual needs.
Described STb gene extraction agent can adopt the reagent of the extracting DNA of various routine, the commercially available acquisition of these reagent.
Described PCR reagent can adopt the PCR reagent of various routine, the commercially available acquisition of this reagent.
Described PCR primer purified reagent can adopt the PCR primer purified reagent of various routine, the commercially available acquisition of this reagent.
Described reference substance comprises wild-type ALDH2 gene masculine control sample, homozygous mutant ALDH2 gene masculine control sample, heterozygous mutant ALDH2 gene masculine control sample.
Described test kit also comprises negative controls, and described negative controls is ddH
2o.
In addition, in described test kit, also comprise working instructions, be convenient to those skilled in the art and use.
Further, a second aspect of the present invention, additionally provides a kind of using method of aforementioned agents box, comprises step:
(1) preparation of DNA sample to be measured: adopt classical genome DNA extracting method or commercial genome extraction test kit, extract genomic dna for subsequent use from sample;
(2) PCR reaction system is configured: DNA sample to be measured, wild-type ALDH2 gene masculine control sample, homozygous mutant ALDH2 gene masculine control sample, heterozygous mutant ALDH2 gene masculine control sample, ddH2O are added the PCR pipe be equipped with containing 2 times of PCR damping fluids, archaeal dna polymerase, dNTP, wild-type and saltant type ALDH2 gene-specific amplification primer pair respectively, and add ddH2O and supply volume, build pipe lid, composition PCR reaction system;
(3) pcr amplification: pcr amplification is carried out to the PCR reaction system of step (2);
(4) PCR primer purifying: purifying is carried out to step (3) gained PCR primer;
(5) digestion with restriction enzyme: the PCR primer after step (4) purifying is digested with TspRI restriction enzyme;
(6) gel electrophoresis: directly agarose gel electrophoresis is carried out to the product after step (5) digestion with restriction enzyme;
(7) interpretation of result: carry out detected result judgement by above-mentioned condition.
Preferably, in step (1), described samples sources is in the saliva of experimenter.
Preferably, in step (2), described primer pair comprises forward primer and reverse primer, and the nucleotide sequence of forward primer, as shown in SEQ ID NO.3, is specially: 5 '-TGTAACCCATAACCCCCAAG-3 '; The nucleotide sequence of reverse primer, as shown in SEQ ID NO.4, is specially: 5 '-AAACACTGATGGCCTCAAGC-3 '.
Except adopting primer of the present invention, the present invention has no particular limits other each component and final concentration thereof in PCR reaction system, and the general composition adopted when those skilled in the art can set up PCR system according to routine and concentration thereof set up PCR reaction system.Template (as genomic dna) for pcr amplification also can adopt the ordinary method of this area to extract and obtain.
A third aspect of the present invention, additionally provides the purposes of aforementioned agents box in preparation ALDH2 gene detection reagent.
Compared with prior art, beneficial effect of the present invention is as follows:
Core innovative point of the present invention is to have found the restriction enzyme site on an ALDH2 gene, saltant type and wild-type can be differentiated by " can being limited property endonuclease digestion ", only need conventional PCR and gel electrophoresis technology just can realize detecting the mutational site of ALDH2 gene.Do not need additionally to buy sequenator, QPCR instrument or chip scanner, detection can be carried out at Molecular Laboratory common in a large number.Meanwhile, compared with other detection method, the present invention also has fast, cheap, stable and feature accurately, has comprehensive advantage.
Sum up, the present invention has following advantage:
(1) as long as basic PCR instrument and electrophoresis equipment just can complete detection, do not need to rely on the equipment such as QPCR instrument, sequenator and chip scanner, be conducive to the universal of detection;
(2) step is simple, and DNA extracting, PCR, endonuclease digestion and gel electrophoresis are all the most basic Protocols in Molecular Biologies, is conducive to the stable and stdn detected;
(3) cost is low;
(4) cycle is short, can complete detection in 4 hours.
Accompanying drawing explanation
The difference schematic diagram of Fig. 1: ALDH2 wild-type and mutated genes.
, in testing sample 1 and 2, there is homozygous mutant ALDH2 gene in Fig. 2: embodiment 2 detected result; Heterozygous mutant ALDH2 gene is there is in sample to be tested 3 and 4; Wild-type ALDH2 gene is there is in testing sample 5 and 6.
Embodiment
Before further describing the specific embodiment of the invention, should be understood that protection scope of the present invention is not limited to following specific specific embodiments; It is also understood that the term used in the embodiment of the present invention is to describe specific specific embodiments, instead of in order to limit the scope of the invention.
When embodiment provides numerical range, should be understood that except non-invention is otherwise noted, between two end points of each numerical range and two end points, any one numerical value all can be selected.Unless otherwise defined, the same meaning that all technology used in the present invention and scientific terminology and those skilled in the art of the present technique understand usually.Except the concrete grammar used in embodiment, equipment, material, according to those skilled in the art to the grasp of prior art and record of the present invention, any method of prior art that is similar with the method described in the embodiment of the present invention, equipment, material or that be equal to, equipment and material can also be used to realize the present invention.
Unless otherwise indicated, disclosed in the present invention experimental technique, detection method, preparation method all adopt the routine techniques of the molecular biology of the art routine, biological chemistry, chromatin Structure and analysis, analytical chemistry, cell cultures, recombinant DNA technology and association area.These technology are existing in existing document improves explanation, specifically can see the MOLECULAR CLONING:A LABORATORY MANUAL such as Sambrook, Second edition, Cold Spring HarborLaboratory Press, 1989and Third edition, 2001; Ausubel etc., CURRENT PROTOCOLS INMOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987and periodic updates; The seriesMETHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATINSTRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODSIN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), AcademicPress, San Diego, 1999; With METHODS IN MOLECULAR BIOLOGY, Vol.119, ChromatinProtocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc.
Embodiment 1 primer and restriction enzyme
The nucleotide sequence of wild-type ALDH2 gene, as shown in SEQ ID NO.1, is specially:
GGCATACACTgAAGTGAAAACT；
And the nucleotide sequence of saltant type ALDH2 gene is as shown in SEQ ID NO.2, be specially:
GGCATACACTaAAGTGAAAACT。
Rectangular boxes has as shown in Figure 1 marked out single base difference of wild-type ALDH2 gene and saltant type ALDH2 gene.
According to wild-type ALDH2 gene and saltant type ALDH2 gene design primer, the position residing in gene structure for mutational site and the feature of sudden change, for the sequence length of primer, position all as the factor of investigation.The present inventor, through repeatedly comparing and optimizing, obtains the primer pair of following can simultaneously increase wild-type and saltant type ALDH2 gene:
Forward primer: 5 '-TGTAACCCATAACCCCCAAG-3 ' SEQ ID NO.3
Reverse primer: 5 '-AAACACTGATGGCCTCAAGC-3 ' SEQ ID NO.4
According to single base difference site of wild-type and saltant type ALDH2 gene, devise a TspRI restriction enzyme, wild-type ALDH2 gene can be digested by described restriction enzyme identification, and restriction enzyme described in saltant type ALDH2 gene pairs is insensitive, therefore digestion can not be identified.
The copy of two ALDH2 genes is had, respectively from male parent and female parent in human body.If two copies are all wild-types, then person under inspection is wild-type; If one is wild-type, one is saltant type, then person under inspection is heterozygous mutant; If be both saltant type, then it is pure and mild saltant type.
First the present invention carries out extracting to the genomic dna of subjects saliva's sample, then the primer pair ALDH2 gene of above-mentioned can simultaneously increase wild-type and saltant type ALDH2 gene is adopted to carry out pcr amplification, after purifying is carried out to gained PCR primer, digest with TspRI restriction enzyme, then postdigestive product is directly carried out electrophoresis.
Can the present invention be by " by the endonuclease digestion " foundation as ALDH2 gene type, so only need to observe the master tape or postdigestive band that whether there is 400bp, can realize somatotype.
If electrophoresis result only has the band of complete an about 400bp, then illustrate that two copies of the ALDH2 gene of experimenter can not, by TspRI digestion with restriction enzyme, be all homozygous mutant ALDH2 gene; If electrophoresis result has two digested bands, then two copies of the ALDH2 gene of experimenter can, by TspRI digestion with restriction enzyme, be all wild-type ALDH2 gene; If electrophoresis result has three bands (wherein having the not digested band of an about 400bp and two digested bands), then the ALDH2 gene of experimenter has a copy can by TspRI digestion with restriction enzyme, one can not, by TspRI digestion with restriction enzyme, be heterozygous mutant ALDH2 gene.
Embodiment 2 test kit detects
By the using method of ALDH2 gene detecting kit of the present invention and test kit, carry out ALDH2 gene to the sample of 3 experimenters and detect, concrete steps are as follows:
1. the preparation of DNA sample to be measured: extract test kit by genome, extracts genomic dna for subsequent use from 3 parts of subjects saliva's samples;
2. configure PCR reaction system:
3.PCR increases: 40 PCR circulations are carried out in 95 DEG C of warm starts after 5 minutes, melting temperature(Tm) 95 DEG C 30 seconds, annealing temperature 58 DEG C 30 seconds, elongating temperature 72 DEG C 40 seconds; After last takes turns PCR, 72 DEG C extend 10 minutes, enter 4 DEG C of preservations;
4.PCR product purification: with paramagnetic particle method, recovery is carried out to PCR primer and purify, and carry out quality inspection;
5. digestion with restriction enzyme: the PCR primer after purifying is digested with TspRI restriction enzyme, reaction system is 50 μ l, and reaction conditions is 65 DEG C, and the reaction times is 2 hours;
6. gel electrophoresis: directly agarose gel electrophoresis is carried out to the product after digestion with restriction enzyme;
7. interpretation of result: the SNP somatotype being carried out ALDH2 gene by the size and number of gel electrophoresis gained band.
The sample that this experiment have employed 6 genotype known (being detected by generation sequencing) is verified, as shown in Figure 2, in Fig. 2, sample 1 and 2 is pure and mild saltant type to result, can not be digested by restriction endonuclease TspRI, be still the master tape of an about 400bp after endonuclease reaction; Sample 3 and 4 is heterozygote, and the copy of half can not by endonuclease digestion, so leave the band of a 400bp; Their second half copy can be become more than 100 by endonuclease digestion and the band of bp more than 200.Due to heterozygote only has the copy of half can be digested, and consider the efficiency of endonuclease reaction, template concentrations can not be too large, cause running digestion products in cementing fruit (the Article 3 band that particularly fragment is slightly little) signal not obvious.But, because can the present invention be by " by the endonuclease digestion " foundation as ALDH2 gene type, so only need to observe the master tape or postdigestive band that whether there is 400bp, can realize somatotype, the 3rd the whether clear this patent that do not affect of band is to genotypic differentiation.Sample 5 and 6 is pure and mild wild-type, and master tape, by complete digestion, is only left two postdigestive digestion products bands.
The above; be only preferred embodiment of the present invention; not to any formal and substantial restriction of the present invention; should be understood that; for those skilled in the art; under the prerequisite not departing from the inventive method, also can make some improvement and supplement, these improve and supplement and also should be considered as protection scope of the present invention.All those skilled in the art, without departing from the spirit and scope of the present invention, a little change made when utilizing disclosed above technology contents, the equivalent variations of modifying and developing, be Equivalent embodiments of the present invention; Meanwhile, all according to substantial technological of the present invention to the change of any equivalent variations that above-described embodiment is done, modify and differentiation, all still belong in the scope of technical scheme of the present invention.
Claims (9)
1. an ALDH2 gene detecting kit, comprises wild-type and saltant type ALDH2 gene-specific amplification primer pair and TspRI restriction enzyme.
2. test kit according to claim 1, is characterized in that, described primer pair comprises forward primer and reverse primer, and the nucleotide sequence of forward primer is as shown in SEQ ID NO.3, and the nucleotide sequence of reverse primer is as shown in SEQ ID NO.4.
3. test kit according to claim 1, is characterized in that, also comprises one or more in STb gene extraction agent, PCR reagent, PCR primer purified reagent, gel electrophoresis reagent and reference substance in described test kit.
4. test kit according to claim 3, is characterized in that, described reference substance comprises wild-type ALDH2 gene masculine control sample, homozygous mutant ALDH2 gene masculine control sample, heterozygous mutant ALDH2 gene masculine control sample.
5. test kit according to claim 3, is characterized in that, described reference substance also comprises negative controls, and described negative controls is ddH
2o.
6. the using method of the test kit according to the arbitrary claim of Claims 1 to 5, comprises step:
(1) preparation of DNA sample to be measured: adopt classical genome DNA extracting method or commercial genome extraction test kit, extract genomic dna for subsequent use from sample;
(2) PCR reaction system is configured: by DNA sample to be measured, wild-type ALDH2 gene masculine control sample, homozygous mutant ALDH2 gene masculine control sample, heterozygous mutant ALDH2 gene masculine control sample, ddH
2o adds the PCR pipe be equipped with containing 2 times of PCR damping fluids, archaeal dna polymerase, dNTP, wild-type and saltant type ALDH2 gene-specific amplification primer pair respectively, and adds ddH
2o supplies volume, builds pipe lid, composition PCR reaction system;
(3) pcr amplification: pcr amplification is carried out to the PCR reaction system of step (2);
(4) PCR primer purifying: purifying is carried out to step (3) gained PCR primer;
(5) digestion with restriction enzyme: the PCR primer after step (4) purifying is digested with TspRI restriction enzyme;
(6) gel electrophoresis: directly agarose gel electrophoresis is carried out to the product after step (5) digestion with restriction enzyme;
(7) interpretation of result: carry out the judgement of ALDH2 genotype according to the detected result of step (6).
7. method according to claim 6, is characterized in that, the samples sources described in step (1) is in the saliva of experimenter.
8. method according to claim 6, is characterized in that, the nucleotide sequence of step (2) forward primer is as shown in SEQ ID NO.3, and the nucleotide sequence of reverse primer is as shown in SEQ ID NO.4; Step (5) described restriction enzyme is TspRI restriction enzyme.
9. the purposes of the test kit according to the arbitrary claim of Claims 1 to 5 in preparation ALDH2 gene detection reagent.
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