CN108531574A - It detects alcohol dehydrogenase ADH1B and ethyl alcohol takes off the primer and method of acetaldehyde dehydrogenase ALDH2 gene pleiomorphisms - Google Patents
It detects alcohol dehydrogenase ADH1B and ethyl alcohol takes off the primer and method of acetaldehyde dehydrogenase ALDH2 gene pleiomorphisms Download PDFInfo
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Abstract
The invention discloses method, primer and kit that the relevant two gene alcohol dehydrogenase ADH1B of alcohol metabolism and ethyl alcohol take off acetaldehyde dehydrogenase ALDH2 polymorphic detections, the primer, kit include the forward and reverse primer and sequencing primer that amplification alcohol dehydrogenase ADH1B and ethyl alcohol take off acetaldehyde dehydrogenase ALDH2 polymorphic sites.The present invention uses Sanger PCR sequencing PCRs, can be used for quickly detecting alcohol dehydrogenase ADH1B using the primer and ethyl alcohol takes off the catastrophe of acetaldehyde dehydrogenase ALDH2 polymorphic sites.
Description
Technical field
The invention belongs to life sciences and biotechnology, more particularly to detect alcohol dehydrogenase ADH1B and ethyl alcohol takes off second
The primer and method of aldehyde dehydrogenase ALDH2 gene pleiomorphisms
Background technology
Alcohol dehydrogenase ADH and ethyl alcohol generate oxidation after taking off acetaldehyde dehydrogenase ALDH2, by oxidation of ethanol at acetaldehyde and
Acetic acid assumes responsibility for most of alcohol metabolism task, is most important alcohol metabolism in human body.
Ethyl alcohol generates acetaldehyde to the first step of ADH systems mainly under ADH effects, and 80% alcohol oxidation is in liver
It is carried out by the approach；Second step, acetaldehyde are generated under the action of ALDH to acetic acid of the body without toxicological effect, blood after drinking
What the acetaldehyde concentration in liquid was mainly determined by ALDH.ADH and ALDH in liver cell are in Coenzyme I (NAD+) presence to alcohol
Normal physiological metabolism plays a significant role jointly, the focus that always people study for many years.But ADH and ALDH contain in human body
It measures limited, when heavy drinking, a large amount of alcohol cannot be metabolized in time, caused drunk.In addition, ADH and ALDH genes are in polymorphic
Property, individual difference is big, causes some to want the people to drink but cannot drink, and drunk people cannot but sober up in time.
ADH is a kind of metalloenzyme, is analyzed through the polymorphic SNP technologies of oligonucleotides, and the human body ADH identified at present is compiled
At least 6 kinds of gene of code, they also have in liver expression highest in the organ-tissues difference journey such as lung, alimentary canal, brain and cornea
The expression of degree.Alcohol dehydrogenase is divided into 3 hypotypes, and wherein the ADH1B in alcohol dehydrogenase 1 (ADH1) is human body alcohol metabolism
In most important isodynamic enzyme, be located at human chromosomal 4q21-25, length is about 15kb, includes 9 exons, aobvious outside the 3rd
G143A point mutation occurs at son, the 47th amino acids is caused to become histidine by arginine, and the allele after change is made to compile
The enzymatic activity of code greatly increases.
ALDH has 4 kinds in human liver, is homotetramer, and encoding gene is positioned at the 12nd chromosome, they are responsible for big
The oxidation of part acetaldehyde.ALDH1 is present in cytoplasm；ALDH2, ALDH3 and ALDH4 are respectively present in different organelles
In.There is wherein ALDH2 genetic polymorphism, allele ALDH2 exons 1s 2 base occurs and replaces, and ALDH2 is caused to lose
It is living.Mutator ALDH2 (2) is dominant, such ALDH2 (2) homozygotes and heterozygous carriers to wild type ALDH2 (1)
ALDH2 it is inactive, be more common in Asia ethnic group.This people's acetaldehyde after drinking alcohol cannot be metabolized in time, caused flushing, headache and disliked
The displeased fast response such as the heart effectively protects ALDH2 (2) carrier without easily indulging in excessive drinking.On the contrary, ALDH2 (1) can be metabolized blood in time
Middle acetaldehyde drinks and relaxation and happiness sense occurs, easily becomes excessive drinking crowd.It is found that 84 SNP on mankind ALDH2 genes altogether at present
Point, but research is concentrated mainly on the sites rs671SNP both at home and abroad.According to the polymorphism of ALDH-2 (rs671), ALDH2 has three
Kind genotype:ALDH2*1/*1 (GG types, wild type), ALDH2*1/*2 (GA types, heterozygous), ALDH2*2/*2 (AA types, mutation
Type).
Alcohol dehydrogenase 1B (alcohol dehydrogenase-1B, ADH1B or ADH-2), aldehyde dehydrogenase 2
(aldehyde dehydrogenase-2, ALDH-2) and smoking, to drink all be the independent hazard factor of the cancer of the esophagus.ALDH-2
(rs671) polymorphism and the generation of myocardial infarction AMI are closely related, and allelic A may be to the generation of young heart infarction
Facilitation
The present invention takes off acetaldehyde dehydrogenase ALDH2 genes using Sanger PCR sequencing PCRs detection alcohol dehydrogenase ADH1B and ethyl alcohol
Polymorphism, and it includes that alcohol dehydrogenase ADH1B and the de- acetaldehyde dehydrogenase ALDH2 genes of ethyl alcohol are more that the primer designed expands respectively
The DNA fragmentation in state property site can get information about alcohol dehydrogenase ADH1B and ethyl alcohol is de- very much by the analysis of sequencing result
The catastrophe of acetaldehyde dehydrogenase ALDH2 gene polymorphism sites, amplimer adds M13 connectors when designing, and uses M13
As sequencing primer, simplifies operating procedure and saved testing cost.
Invention content
The present invention provides detection alcohol dehydrogenase ADH1B and ethyl alcohol takes off acetaldehyde dehydrogenase ALDH2 gene polymorphism sites
Primer can be used for quickly detecting alcohol dehydrogenase ADH1B and ethyl alcohol take off acetaldehyde dehydrogenase ALDH2 bases using Sanger PCR sequencing PCRs
The case where because of polymorphic site.
The purpose of the present invention is to provide detection alcohol dehydrogenase ADH1B and the de- acetaldehyde dehydrogenase ALDH2 genes of ethyl alcohol are more
The primer of state property, including：Amplification covering detection alcohol dehydrogenase ADH1B and ethyl alcohol take off acetaldehyde dehydrogenase ALDH2 gene pleiomorphisms
The forward and reverse primer in site, base sequence are：
ADH1B-F：TGTAAAACGACGGCCAGTGGAATAGTAGGGATTAGTAGC；
ADH1B-R：AACAGCTATGACCATGCTCCTGAAGTCCTGGCT；
ALDH2-F：TGTAAAACGACGGCCAGTCATAACCCCCAAGAGTGATTTC；
ALDH2-R：AACAGCTATGACCATGAGAGCAGAGGCTGGGTCTTTAC；
Primer further includes a pair of of sequencing primer, and base sequence is：
M13F：TGTAAAACGACGGCCAGT
M13R：AACAGCTATGACCATG.
Further, the concentration ratio of the forward and reverse primer is：ADH1B-F:ADH1B-R=1:1, ALDH2-F:
ALDH2-R=1:1.
Further, the concentration ratio of the sequencing primer is：M13F:M13R=1:1.
It is a further object to provide detection alcohol dehydrogenase ADH1B and the de- acetaldehyde dehydrogenase ALDH2 of ethyl alcohol are more
The kit in state property site, which is characterized in that the kit includes：
(1) sample DNA extraction agent；
(2) detection architecture PCR reaction solution；Detection alcohol dehydrogenase ADH1B is covered including amplification and ethyl alcohol takes off acetaldehyde-dehydrogenase
The forward and reverse primer of enzyme ALDH2 gene polymorphism sites, base sequence are：
ADH1B-F：TGTAAAACGACGGCCAGTGGAATAGTAGGGATTAGTAGC；
ADH1B-R：AACAGCTATGACCATGCTCCTGAAGTCCTGGCT；
ALDH2-F：TGTAAAACGACGGCCAGTCATAACCCCCAAGAGTGATTTC；
ALDH2-R：AACAGCTATGACCATGAGAGCAGAGGCTGGGTCTTTAC；
(3) system reaction solution is sequenced, including a pair of of sequencing primer, base sequence are：
M13F：TGTAAAACGACGGCCAGT
M13R：AACAGCTATGACCATG；
(4) positive reference substance, negative controls and blank control product.
Further, the detection architecture PCR reaction solution further includes 2 × PCR Buffer；dNTPs；KOD FX DNA
Polymerase。
Further, the sequencing system reaction solution further include sequencing refined solution, EDTA, absolute ethyl alcohol, 75% ethyl alcohol,
HIDI and Bigdye Terminator V3.1.The sequencing refined solution includes shrimp alkaline phosphotase and exonuclease I.
The present invention also aims to provide a kind of the detection alcohol dehydrogenase ADH1B and the de- acetaldehyde of ethyl alcohol of non-diagnostic purpose
The method of dehydrogenase A LDH2 gene pleiomorphisms, which is characterized in that include the following steps：
(1) sample DNA is extracted；
(2) amplimer ADH1B-F/ADH1B-R, ALDH2-F/ALDH2-R are utilized, the DNA in (1) is expanded,
Obtain the amplified production of lid detection ADH1B and ALDH2 gene pleiomorphisms；
(3) sequencing primer M13F and M13R are utilized, forward and reverse sequencing is carried out to the amplified production in (2), obtains institute
State the gene order of amplified production；
(4) gene order in (3) is compared with wild type ADH1B and ALDH gene order, determines mutational site
It whether there is；
Wherein, amplification covering detection alcohol dehydrogenase ADH1B and ethyl alcohol take off acetaldehyde dehydrogenase ALDH2 gene polymorphism sites
The base sequence of forward and reverse primer be：
ADH1B-F：TGTAAAACGACGGCCAGTGGAATAGTAGGGATTAGTAGC；
ADH1B-R：AACAGCTATGACCATGCTCCTGAAGTCCTGGCT；
ALDH2-F：TGTAAAACGACGGCCAGTCATAACCCCCAAGAGTGATTTC；
ALDH2-R：AACAGCTATGACCATGAGAGCAGAGGCTGGGTCTTTAC；
The base sequence of sequencing primer is：
M13F：TGTAAAACGACGGCCAGT
M13R：AACAGCTATGACCATG.
The present invention devises amplification alcohol dehydrogenase ADH1B and ethyl alcohol take off acetaldehyde dehydrogenase ALDH2 polymorphic sites just,
Reverse primer, and innovative added one section of long 18bp's respectively in PCR amplification sense primer front end and downstream primer front end
The M13R primer sequences of M13F primer sequences and long 16bp.Introduced M13F can all be taken by expanding later product both ends in this way
And M13R primer sequences, when then carrying out sequencing reaction, all amplified productions can use unified M13F and M13R
Primer can be carried out forward and reverse sequencing.PCR amplification is carried out to inspection sample, using Sanger PCR sequencing PCRs, to PCR product into
The forward and reverse sequencing reaction amplification of row, after purification denaturation, direct Sequencing can comprehensively detect alcohol dehydrogenase ADH1B and ethyl alcohol is de-
The catastrophe of acetaldehyde dehydrogenase ALDH2 polymorphic sites.Concentration, annealing temperature by adjusting forward and reverse primer etc. react item
Part can make amplification efficiency reach best.
Advantageous effect：Using extension primer of the present invention and sequencing primer, can expand alcohol dehydrogenase ADH1B and
Ethyl alcohol takes off acetaldehyde dehydrogenase ALDH2 polymorphic sites can get information about alcohol dehydrogenase very much by the analysis of sequencing result
ADH1B and ethyl alcohol take off acetaldehyde dehydrogenase ALDH2 polymorphic site gene mutation situations, not by the diversified shadow of gene mutation
It rings, all mutational sites to be detected can be covered；With very high specificity and accuracy；Purpose base is expanded using PCR method
Because and be sequenced detect its gene pleiomorphism, have many advantages, such as high sensitivity, it is easy to operate, at low cost.
Description of the drawings
The sequencing result of Fig. 1 samples 1ADH1B.
The testing result figure of Fig. 2 samples 1ALDH2.
The sequencing result of Fig. 3 samples 2ADH1B.
The testing result figure of Fig. 4 samples 2ALDH2.
The sequencing result of Fig. 5 samples 3ADH1B.
The testing result figure of Fig. 6 samples 3ALDH2.
Specific implementation mode
With reference to specific embodiments and the drawings, the present invention is further explained.It should be noted that not specified in embodiment
Normal condition and method, usually according to fields experimenter routinely use method：For example, Ao Sibai and James Kingston chief editor
's《Fine works molecular biology experiment guide》Fourth edition, or according to the step and condition proposed by manufacturer.
Embodiment 1
It detects alcohol dehydrogenase ADH1B and ethyl alcohol takes off the primer of acetaldehyde dehydrogenase ALDH2 polymorphic sites, including：Amplification
It detects alcohol dehydrogenase ADH1B and ethyl alcohol takes off the forward and reverse primer of acetaldehyde dehydrogenase ALDH2 polymorphic sites；The extension is drawn
The base sequence of object is：
| ADH1B-F | TGTAAAACGACGGCCAGTGGAATAGTAGGGATTAGTAGC |
| ADH1B-R | AACAGCTATGACCATGCTCCTGAAGTCCTGGCT |
| ALDH2-F | TGTAAAACGACGGCCAGTCATAACCCCCAAGAGTGATTTC |
| ALDH2-R | AACAGCTATGACCATGAGAGCAGAGGCTGGGTCTTTAC |
The primer further includes sequencing primer, and base sequence is
M13F：TGTAAAACGACGGCCAGT
M13R：AACAGCTATGACCATG
In the detection, acetaldehyde dehydrogenase is taken off to alcohol dehydrogenase ADH1B and ethyl alcohol first with above-mentioned forward and reverse primer
The DNA fragmentation of ALDH2 is expanded, and is obtained amplified production, is then sequenced, is obtained to amplified production using above-mentioned sequencing primer
Obtain the gene order of amplified production.
It detects alcohol dehydrogenase ADH1B and ethyl alcohol takes off the kit in acetaldehyde dehydrogenase ALDH2 polymorphic sites site, packet
It includes：Tissue DNA extraction agent box (such as extracts kit using Tiangeng biology)；Absolute ethyl alcohol；Detection architecture PCR reactions
Liquid, sequencing system reaction solution, positive reference substance, negative controls and blank control product, wherein
Detection architecture PCR reaction solution includes：2×PCR Buffer；2mM dNTPs；KOD FX DNA Polymerase
(1U/μl)；The upstream and downstream primer of testing goal gene.
Sequencing system includes：Sequencing refined solution, EDTA (125mmol), absolute ethyl alcohol, 75% ethyl alcohol, HID I (are highly gone
Formamide), sequencing primer：M13F (3.2 μm) and M13R (3.2 μm) and Bigd ye Terminator V3.1 (purchases
Buy from Applied Biosystems companies of the U.S.), wherein sequencing refined solution includes shrimp alkaline phosphotase (SAP) 0.6U and nucleic acid
Excision enzyme I (EXONI) 1.2U.
Detection architecture PCR reaction solution is formulated as follows：
Positive reference substance：Solution containing ADH and ALDH sequences.
Negative controls：Solution without ADH and ALDH sequences.
Blank control product：2 μ l physiological saline are not added with any substance.
Embodiment 2
The operating process of blood DNA extraction agent box (Tiangeng biology)：
(1) genomic DNA in blood is extracted:
1) it extracts 500uL blood and 1000uL erythrocyte cracked liquids is added, overturn mixing, be placed at room temperature for 5 minutes, during which run again
Mixing is several times.3000rpm centrifuges 5min, sucks supernatant, leaves leukocyte cell pellet, adds 200uL buffer solution GA, vibrates to thorough
Bottom mixing.
2) 20 μ l Proteinase K Solutions, mixing is added.
3) 200 μ l buffer solution GB are added, fully reverse mixing, 70 DEG C are placed 10 minutes, and solution strains limpid, brief centrifugation
To remove the droplet of cap wall.
4) 200 μ l absolute ethyl alcohols are added, fully vibrate mixing 15 seconds, at this time it is possible that flocculent deposit, brief centrifugation
To remove the droplet of cap wall.
5) previous step acquired solution and flocculent deposit be all added in an adsorption column CB3 (adsorption column is put into collecting pipe
In), 12,000rpm (13,400 × g) are centrifuged 30 seconds, outwell waste liquid, adsorption column CB3 is put back in collecting pipe.
6) 500 μ l buffer solutions GD (please first checked whether before use and absolute ethyl alcohol has been added) are added into adsorption column CB3,
12,000rpm (13,400 × g) are centrifuged 30 seconds, outwell waste liquid, adsorption column CB3 is put into collecting pipe.
7) 700 μ l rinsing liquids PW (please first checked whether before use and absolute ethyl alcohol has been added) are added into adsorption column CB3,
12,000rpm (13,400 × g) are centrifuged 30 seconds, outwell waste liquid, adsorption column CB3 is put into collecting pipe.
8) 500 μ l rinsing liquids PW, 12,000rpm (13,400 × g) centrifugation 30 seconds is added into adsorption column CB3, outwells useless
Liquid.
9) adsorption column CB3 is put back in collecting pipe, 12,000rpm (13,400 × g) are centrifuged 2 minutes, outwell waste liquid.It will inhale
Attached column CB3, which is placed in, to be placed at room temperature for several minutes, thoroughly to dry rinsing liquid remaining in sorbing material.
10) adsorption column CB3 is transferred in a clean centrifuge tube, 100 μ l is vacantly added dropwise to the intermediate position of adsorbed film
Elution buffer TE is placed at room temperature for 2-5 minutes, and 12,000rpm (13,400 × g) are centrifuged 2 minutes, and solution is collected into centrifuge tube
In.
(2) reagent configures：Each X μ l of detection architecture PCR reaction solution are configured by detection person-portion number, per 18 μ l packing of person-portion：
X=18 μ l reaction solutions × (+1 part of blank control of n parts of+1 part of sample+1 part of positive control negative controls)
N is detection number of samples.
(3) it is loaded：Each 2 μ l DNA into PCR reaction solution；Positive control and negative control directly add 2 μ l positive reference substances
And negative controls；Blank control adds 2 μ l physiological saline or is not added with any substance.
(4) it expands：Detection carries out on Standard PCR instrument, can include ABI veriti (U.S. Applied with instrument
Biosystems companies) etc..Reaction condition is as follows：
(5) sanger is sequenced：
Take 9 μ l PCR products and 2 μ l purification systems.It is purified according to following procedure：
By 1 μ l purified products respectively with sequencing primer：M13-F (3.2 μm) and M13-R (3.2 μm) according to following system into
Row mixing：
Sequencing reaction program：
Precipitate link：
The EDTA of 2 μ l 125mmol is added into the product for completing sequencing reaction, stands 5min；The anhydrous second of 15ml is added
Alcohol, whirlpool mixing；3700rpm centrifuges 30min；It is inverted centrifugation 15sec, 50ml70% ethyl alcohol, whirlpool mixing is added；3700rpm
Centrifuge 15min；It is inverted centrifugation 15sec, is placed on 95 DEG C of metal baths；Denatured test is carried out after 10 μ l HIDI are added.It is denaturalized journey
Sequence：
After denaturation program, upper sequenator (ABI3730) sequencing.
(7) result judges：Sequencing result is compared with wild-type reference sequence, according to practical catastrophe to result
It is reported.
Embodiment 3
3 parts of clinical whole blood sample is taken, the relevant two gene alcohol dehydrogenases ADH1B of 2 parts of sample alcohol metabolisms is detected
The polymorphic implementations of acetaldehyde dehydrogenase ALDH2 are taken off with ethyl alcohol.By 2 the method for embodiment extraction genome, reagent preparation and examine
It surveys.2 μ l in detection architecture PCR reaction solution are added in every part of sample.The positive is done simultaneously, negative, each portion of blank control.With common
PCR instrument detects, and the time is 160 minutes.
From testing result as can be seen that primer of the present invention is capable of detecting when that alcohol dehydrogenase ADH1B and ethyl alcohol take off second
Aldehyde dehydrogenase ALDH2, and sequencing result entirely accurate.Primer of the present invention can accurately amplify alcohol dehydrogenase
ADH1B and ethyl alcohol take off acetaldehyde dehydrogenase ALDH2 genes, either wild type or saltant type.The detection of positive sample is shown
Primer of the present invention and method and kit are capable of detecting when that alcohol dehydrogenase ADH1B and ethyl alcohol take off acetaldehyde dehydrogenase ALDH2
Gene mutation.
Sequence table
<110>ADICON Clinical Laboratories, Inc.
<120>It detects alcohol dehydrogenase ADH1B and ethyl alcohol takes off the primer and method of acetaldehyde dehydrogenase ALDH2 gene pleiomorphisms
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 39
<212> DNA
<213>Artificial sequence ()
<400> 1
tgtaaaacga cggccagtgg aatagtaggg attagtagc 39
<210> 2
<211> 33
<212> DNA
<213>Artificial sequence ()
<400> 2
aacagctatg accatgctcc tgaagtcctg gct 33
<210> 3
<211> 40
<212> DNA
<213>Artificial sequence ()
<400> 3
tgtaaaacga cggccagtca taacccccaa gagtgatttc 40
<210> 4
<211> 38
<212> DNA
<213>Artificial sequence ()
<400> 4
aacagctatg accatgagag cagaggctgg gtctttac 38
<210> 5
<211> 18
<212> DNA
<213>Artificial sequence ()
<400> 5
tgtaaaacga cggccagt 18
<210> 6
<211> 16
<212> DNA
<213>Artificial sequence ()
<400> 6
aacagctatg accatg 16
Claims (5)
1. detecting alcohol dehydrogenase ADH1B and ethyl alcohol taking off the primer of acetaldehyde dehydrogenase ALDH2 gene pleiomorphisms, which is characterized in that
Including：Amplification covering detection alcohol dehydrogenase ADH1B and ethyl alcohol take off the positive and negative of acetaldehyde dehydrogenase ALDH2 gene polymorphism sites
To primer, base sequence is：
ADH1B-F：TGTAAAACGACGGCCAGTGGAATAGTAGGGATTAGTAGC；
ADH1B-R：AACAGCTATGACCATGCTCCTGAAGTCCTGGCT；
ALDH2-F：TGTAAAACGACGGCCAGTCATAACCCCCAAGAGTGATTTC；
ALDH2-R：AACAGCTATGACCATGAGAGCAGAGGCTGGGTCTTTAC；
Primer further includes a pair of of sequencing primer, and base sequence is：
M13F：TGTAAAACGACGGCCAGT
M13R：AACAGCTATGACCATG.
2. primer as described in claim 1, which is characterized in that the concentration ratio of the forward and reverse primer is：ADH1B-F:
ADH1B-R=1:1,ALDH2-F:ALDH2-R=1:1.
3. primer as described in claim 1, which is characterized in that the concentration ratio of the sequencing primer is：M13F:M13R=1:1.
4. detecting alcohol dehydrogenase ADH1B and ethyl alcohol taking off the kit of acetaldehyde dehydrogenase ALDH2 polymorphic sites, feature exists
In the kit includes：
(1) sample DNA extraction agent；
(2) detection architecture PCR reaction solution；Detection alcohol dehydrogenase ADH1B is covered including amplification and ethyl alcohol takes off acetaldehyde dehydrogenase
The forward and reverse primer of ALDH2 gene polymorphism sites, base sequence are：
ADH1B-F：TGTAAAACGACGGCCAGTGGAATAGTAGGGATTAGTAGC；
ADH1B-R：AACAGCTATGACCATGCTCCTGAAGTCCTGGCT；
ALDH2-F：TGTAAAACGACGGCCAGTCATAACCCCCAAGAGTGATTTC；
ALDH2-R：AACAGCTATGACCATGAGAGCAGAGGCTGGGTCTTTAC；
(3) system reaction solution is sequenced, including a pair of of sequencing primer, base sequence are：
M13F：TGTAAAACGACGGCCAGT
M13R：AACAGCTATGACCATG；
(4) positive reference substance, negative controls and blank control product.
5. the detection alcohol dehydrogenase ADH1B and ethyl alcohol of a kind of non-diagnostic purpose take off acetaldehyde dehydrogenase ALDH2 gene pleiomorphisms
Method, which is characterized in that include the following steps：
(1) sample DNA is extracted；
(2) DNA in (1) is expanded respectively using amplimer ADH1B-F/ADH1B-R, ALDH2-F/ALDH2-R, is obtained
Alcohol dehydrogenase ADH1B must be detected and ethyl alcohol takes off the amplified production of acetaldehyde dehydrogenase ALDH2 gene pleiomorphisms；
(3) forward and reverse sequencing carried out respectively to the amplified production in (2) using sequencing primer M13F and M13R, described in acquisition
The gene order of amplified production；
(4) gene order in (3) is compared with wild type ADH1B and ALDH gene order, whether determines mutational site
In the presence of；
Wherein, amplification covering detection alcohol dehydrogenase ADH1B and ethyl alcohol take off acetaldehyde dehydrogenase ALDH2 gene polymorphism sites
The base sequence of forward and reverse primer is：
ADH1B-F：TGTAAAACGACGGCCAGTGGAATAGTAGGGATTAGTAGC；
ADH1B-R：AACAGCTATGACCATGCTCCTGAAGTCCTGGCT；
ALDH2-F：TGTAAAACGACGGCCAGTCATAACCCCCAAGAGTGATTTC；
ALDH2-R：AACAGCTATGACCATGAGAGCAGAGGCTGGGTCTTTAC；
The base sequence of sequencing primer is：
M13F：TGTAAAACGACGGCCAGT
M13R：AACAGCTATGACCATG.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109628577A (en) * | 2019-01-07 | 2019-04-16 | 杭州艾迪康医学检验中心有限公司 | Detect the primer and method of coronary sclerosis correlation mononucleotide polymorphism site |
| CN114409731A (en) * | 2022-01-05 | 2022-04-29 | 浙江省农业科学院 | Two polypeptides having activity of activating alcohol dehydrogenase and acetaldehyde dehydrogenase simultaneously |
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