CN102251023A - Method, primer and TaqMan-MGB probe for detecting mutation of alcohol metabolism-related gene - Google Patents
Method, primer and TaqMan-MGB probe for detecting mutation of alcohol metabolism-related gene Download PDFInfo
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Abstract
The invention discloses a method, a primer and a TaqMan-MGB probe for detecting mutation of an alcohol metabolism-related gene. The prime for detecting mutation of the alcohol metabolism-related gene is composed of eight DNA sequences of SEQ ID:1-SEQ ID:8 in a sequence table. The TaqMan-MGB probe is eight DNA sequences of SEQ ID:9-SEQ ID:16 in the sequence table, 5'-terminuses of the DNA sequences are marked with reporter fluorescence groups with different luminous colors, and 3'-terminuses of the DNA sequences are marked with non-fluorescent quenchers and are connected with dehydrocyclopyrroindole tripetide. The invention provides a new approach with rapidness, reliability and accuracy for detecting, testing, analyzing, and assessing alcohol metabolism, thereby providing a theoretical base for screening high sensitive individuals and reducing alcohol-related disease generation through adopting preventive measures.
Description
Technical field
The present invention relates to mutating alkali yl be carried out the primer and the probe of qualitative and quantitative analysis with molecular biology method.Particularly relate to the primer and the TaqMan-MGB probe that the alcohol metabolism associated gene mutation are carried out qualitative and quantitative analysis with real-time fluorescence quantitative PCR (real-time fluorescent quantificationPCR) technology.
Background technology
Drinking to cause multiple disease, comprises chronic gastritis, toxic hepatitis, myocardial hypertrophy, lithangiuria, irreversible cerebral lesion and alcoholism, mental disorder and personality change etc.The Health hazard of drinking mainly causes by alcohol (ethanol) and mesostate acetaldehyde thereof, both in vivo the effective dose level and action time length remove and the alcohol intake, outside the frequency dependence of drinking, also relevant with intravital metabolic enzyme activity.
Generally speaking, ethanol is restricted by ethanol dehydrogenase (ADH), acetaldehyde dehydrogenase (ALDH) and Cytochrome P450 (CYP2E1) in the liver mainly in the body metabolism process.Ethanol changes into acetaldehyde in liver, acetaldehyde reoxidizes and is acetate, resolves into carbonic acid gas and water excretes.
Find to have the ADH (ADH1-ADH7) of 7 kinds of polymorphisms among the crowd, wherein ADH1B and ADH1C play a very important role in the alcohol metabolism process.
ADH1B is produced by the ADH1B genes encoding, and different genotype influence the activity of ADH1B, thereby acetaldehyde concentration in the body is exerted an influence.There is the A/G polymorphism in the ADH1B gene.G allelotrope is wild-type, and the activity of carrying ADH1B in this allelic people colony is lower with respect to carrying the allelic crowd of A.A allelotrope makes ADH1B have higher activity, carries this allelic people and can rapidly the alcohol catalysis reaction that absorbs be formed acetaldehyde.The genotypic individual enzyme of AA is lived very high, belongs to fast metabolic pattern, ethanol can be changed into acetaldehyde soon.
The γ subunit of a kind of alcoholdehydrogenase ADH of ADH1C genes encoding.Ethanol arrives liver cell with blood circulation, and katabolism is activated.There is an A/G polymorphism in the ADH1C gene, and this polymorphism can cause the dimer ADH of its coding to have different activity, and then causes the speed difference of Different Individual to alcohol metabolism.Wherein the allelic ADH1C enzymic activity of A type is about allelic 2.5 times of G type.Its ADH1C enzyme of AA genotype has very high catalytic activity, belongs to fast metabolic pattern, can acceleration bodies in the metabolism of alcohol.
The ALDH that has 12 kinds of different genes coding in human organ and the tissue at least, common have four kinds of ALDH1-ALDH4, is the main isozyme in the human liver.In 12 kinds of ALDH, have only ALDH2 to show genetic polymorphism.ALDH2 is positioned at plastosome, and ALDH1, ALDH3, ALDH4 is positioned at cytosol.Metabolic process in the ALDH2 catalysis alcohol metabolism process from acetaldehyde to acetate.ALDH2 genotype and alcohol user are to alcoholic acid susceptibility, tolerance, reaction after drinking, drinking behavior and diseases induced closely related.This pleomorphism site forms the G/A polymorphism, can cause a glutamic acid rotating in the ALDH2 aminoacid sequence to turn to Methionin, causes the change of ALDH2 enzymic activity.ALDH2 is four polyacetylene compound enzymes, and sudden change has influenced the stability of tetramer structure, and then can influence the activity of enzyme, and then the efficient of alcohol metabolism process is caused different influences.A allelotrope causes the rapid decline of ALDH2 enzymic activity, and G allelotrope then keeps its normal activity
Cytochrome P450 family member CYP2E1 is relevant with functions such as drug metabolism and lipid are synthetic.Its and chemical substance external source endogenous to some has metabolic function, for example alcohol, acetone or the like.CYP2E1 is one of key enzyme that plays a major role in the alcohol metabolism, and it is subjected to alcohol induced and participates in the alcoholic acid metabolism.There is a C/G polymorphism in this gene, and this polymorphic position is in the transcriptional regulatory zone of CYP2E1, and C allelotrope may directly influence the expression of downstream exon, thereby influences the activity of CYP2E1 enzyme, and individual metabolic capacity to materials such as alcohol is changed.It has related with the susceptibility of diseases such as liver injury, liver cirrhosis and kinds of tumors.
By detecting the polymorphism characteristics of ADH1B, ADH1C, ALDH2 and these 4 genes of CYP2E1, can learn the active height of corresponding enzymes metabolism, judge individual alcohol metabolism ability and characteristics, thereby for screening high-order sensitive individual and taking preventive measures to reduce diseases related the providing fundamental basis of ethanol.
At present, the method for having reported that is used for the alcohol metabolism associated gene mutation mainly comprises PCR directly order-checking and qualitative PCR etc., and these method ubiquity sensitivity are low, and specificity is not high, wastes time and energy and easy shortcoming such as pollution, makes application be subjected to certain limitation.
The real-time fluorescence quantitative PCR technology, be meant in the PCR reaction system and add a specificity fluorescent probe in a pair of primer of adding, probe only with the template specific combination, its binding site utilizes the fluorescent signal accumulation whole PCR process of monitoring in real time between two primers.TaqMan fluorescent probe at present commonly used is an oligonucleotide, and its 5 ' end is marked with the report fluorophor, as FAM, and VIC etc., 3 ' end is marked with the cancellation fluorophor, as TAMRA etc.When probe is complete, the fluorescent signal that reporter group sends is absorbed by quenching group, so detect less than fluorescence, when probe and target sequence pairing, 5 ' → 3 ' 5 prime excision enzyme activity of PCR extension polysaccharase is cut degraded with the probe enzyme, be that the report fluorophor separates with the cancellation fluorophor, thereby fluorescence detecting system can receive fluorescent signal.Every through a PCR circulation, fluorescent signal is also the same with the purpose fragment, the process that has a sync index to increase, detect first order fluorescence intensity after each loop ends, after entire reaction finishes, just can obtain an amplification curve, can obtain a typical curve, can carry out quantitative analysis to unknown template according to the amplification curve of this typical curve and unknown template by the amplification curve of concentration known standard model.But in fact, probe is long to make that two ends group distance is far away, can cause fluorescent quenching not thorough, and fluorophor also can produce the fluorescence of different wave length, can make that all background is higher.
At the halfway problem of TaqMan fluorescence probe cancellation, American AB I company had released a kind of new TaqMan-MGB probe (TaqMan Minor Groove Binder Probe in 2000, the TaqMan-MGB probe), the TaqMa-MGB probe is that the widow who has a minor groove binders group examines the former times acid probe, principle of work is identical with the TaqMan probe, the report fluorophor is connected 5 ' end of probe (as FAM, VIC etc.), 3 ' end is marked with quenching group, different is that quenching group is non-luminous quenching group (Non-FluorescentQuenoher, NFQ), quenching group absorbs behind the energy of reporter group not luminous, greatly reduces the interference of background signal.In addition, 3 ' end of MGB probe has also connected a dihydro ring annulated indole porphyrin-tripeptides (dehydrocyclopyrroindole tripetide, DPI3), DPI3 is collapsible have been advanced in the ditch that is formed by the acid of probe end 5-6bp nuclear battalion.It is capable that DPI3 is crescent, with the same spiral of shallow dark ditch of B-form DNA spiral, mainly stable by Van der Waals force.The TaqMan-MGB probe has shown stronger sequence-specific.Can stablize the hybridization of probe and template greatly, rising probe Tm value makes short probe can reach higher Tm value one equally and to lack the distance of the fluorescence report group of probe and quenching group nearer, the cancellation better effects if, fluorescence background is lower, makes signal to noise ratio higher.
In a word, with traditional TaqMan probe comparison, the TaqMan-MGB probe has following advantage: MGB to increase probe melting temperature (Tm), can make probe shorter like this, especially be fit to be rich in the sequence of A/T, and the Tm value difference that improves between pairing and non-matching template is different, can carry out more multiple PCR; Improve signal to noise ratio and since probe 3 ' quenching group of end is non-luminous fluorophor, and more approaching in the spatial position with reporter group, and result of experiment is more accurate, and resolving power is higher; And experimental procedure is simple, and the stability of hybridization improves greatly, and repeatability improves greatly.
Still be not specifically designed at present the real-time fluorescence quantitative PCR detection technique that detects the alcohol metabolism associated gene mutation.
Summary of the invention
The purpose of this invention is to provide the primer that carries out qualitative and quantitative analysis at the skin aging associated gene mutation.
For solving the problems of the technologies described above, the present invention takes following technical scheme:
Be used to detect the primer of ADH1B sudden change, right by the primer that SEQ ID NO:1 in the sequence table and SEQ ID NO:2 form.
Be used to detect the primer of ADH1C sudden change, right by the primer that SEQ ID NO:3 in the sequence table and SEQ ID NO:4 form.
Be used to detect the primer of ALDH2 sudden change, right by the primer that SEQ ID NO:5 in the sequence table and SEQ ID NO:6 form.
Be used to detect the primer of CYP2E1 sudden change, right by the primer that SEQ ID NO:7 in the sequence table and SEQ ID NO:8 form.
The present invention also provides the TaqMan-MGB probe that carries out qualitative and quantitative analysis at the skin health associated gene mutation.
For solving the problems of the technologies described above, the present invention takes following technical scheme:
Being used to detect the ADH1B TaqMan-MGB probe of sudden change, is the dna sequence dna of SEQ ID NO:9 and SEQID NO:10 in the sequence table.
Being used to detect the ADH1C TaqMan-MGB probe of sudden change, is the dna sequence dna of SEQ ID NO:11 and SEQID NO:12 in the sequence table.
Being used to detect the ALDH2 TaqMan-MGB probe of sudden change, is the dna sequence dna of SEQ ID NO:13 and SEQID NO:14 in the sequence table.
Being used to detect the CYP2E1 TaqMan-MGB probe of sudden change, is the dna sequence dna of SEQ ID NO:15 and SEQ ID NO:16 in the sequence table.
Embodiment
The present invention is described further in conjunction with specific embodiments.Should be understood that these embodiment only are used for illustration purpose, and need not limit the scope of the invention.
Embodiment 1, the primer that is used for the detection of alcohol metabolism associated gene mutation and the design of TaqMan-MGB probe
Mutational site according to gene order, with Primer Express Software 2.0 software design PCR primers, reverse complementary sequence according to wild type gene and mutated genes designs 2 TaqMan-MGB probes simultaneously, 5 ' end difference mark report fluorogene FAM (red fluorescence) and VIC (green fluorescence) with 2 TaqMan-MGB probes, the not luminous quenching group of 3 ' end mark, and connect DIP3.Primer and TaqMan-MGB probe sequence are:
Detect the primer of ADH1B sudden change:
5’-agagtgttggagaaggggtgacta-3’
5’-aaatgtatttgagggttttgctac-3’
Detect the TaqMan-MGB probe of ADH1B sudden change:
5’-VIC-gtcatctgtgtgacagattcc-MGB-3’
5’-FAM-gtcatctgtgcgacagattcc-MGB-3’
Detect the primer of ADH1C sudden change:
5’-aaagtttagaaaaagtgctgcatt-3’
5’-tagcagagaatgaaaaggtggaag-3’
Detect the TaqMan-MGB probe of ADH1C sudden change:
5’-VIC-aaaaggtaaaacatttgttatt-MGB-3’
5’-FAM-aaaaggtaaaatatttgttatt-MGB-3’
Detect the primer of ALDH2 sudden change:
5’-ctgctggtggctcggagcctgctg-3’
5’-aaagacgttagagcaaagtttaat-3’
Detect the TaqMan-MGB probe of ALDH2 sudden change:
5’-VIC-gttttcactttagtgtatgcc-MGB-3’
5’-FAM-gttttcacttcagtgtatgcc-MGB-3’
Detect the primer of CYP2E1 sudden change:
5’-agtgatttggctggattgtaaatg-3’
5’-ctgccgcctcctcctctctttcct-3’
Detect the TaqMan-MGB probe of CYP2E1 sudden change:
5’-VIC-ttcaggagaggtgcagtgtta-MGB-3’
5’-FAM-ttcaggagagctgcagtgtta-MGB-3’
The detection of embodiment 2, alcohol metabolism associated gene mutation
1) sample genomic dna obtains
DNA with in the lysis method extraction oral mucosa cast-off cells is suspended in the 1ml physiological saline the centrifugal 10min of 2000 * g; Abandon supernatant, every pipe adds 1ml physiological saline repeated washing once, the more centrifugal 10min of 2000 * g; Abandon supernatant, every pipe add 400 μ l cell lysis buffer solution (10mmol/L Tris-HCl, PH 8.0; 0.1mol EDTA, PH 8.0; 0.5%SDS), be placed in 50 ℃ of water-baths incubation 30 minutes; Be cooled to room temperature then, add isopyknic phenol one chloroform one primary isoamyl alcohol (volume ratio 25: 24: 1), mixing, the centrifugal 10min of 5000 * g; Shift the upper strata water in another Eppendorf pipe, repeat extracting once; Shift the upper strata water again in another Eppendorf pipe, add the 3M NaAc (pH5.2) of 1/10 volume, mixing; The 95% cold ethanol that adds 2.5 times of volumes, mixing ,-20 ℃ of deposit D NA 30 minutes; Centrifugal 15 minutes of 10000 * g abandons supernatant; Add the cold ethanol washing and precipitating of 1ml70%, centrifugal minute of 10000 * g abandons supernatant; The oven dry in 30 minutes of 37 ℃ of incubators; DNA precipitation is dissolved in the 20 μ l TE damping fluids, adds 1 μ l RNA enzyme, 37 ℃ 30 minutes, place-20 ℃ of preservations.
2) pcr amplification
The DNA that extracts with step 1) is a template, under the guiding of embodiment 1 described primer and TaqMan-MGB probe, carry out pcr amplification with ABI 7900 real-time fluorescence quantitative PCR instrument (u.s.a. applied biosystem company), and finish and analyze by allelotrope identification experiment operation steps, promptly read allelotrope identification experiment pcr amplification front signal successively, carry out amplification program and read, analysing amplified back signal.Wherein, the PCR reaction system is: the general pcr amplification premix of 2xTaqMan reagent (available from u.s.a. applied biosystem company), each 900nM of primer, fluorescently-labeled 2 each 200nM of TaqMan-MGB probe, 150ng DNA.The PCR reaction conditions is: earlier 95 ℃ 10 minutes; Then 95 ℃ 15 seconds, 60 ℃ 1 minute, totally 40 circulations.
3) obtain a result
Analyze experimental result with SDS software (u.s.a. applied biosystem company).This experiment is got 3 experimenter's samples altogether and is experimentized, and final genotype distributes and sees the following form.
Sequence table
＜110〉associating gene biological technology (Shanghai) Co., Ltd.
＜120〉method and the primer and the TaqMan-MGB probe of detection alcohol metabolism associated gene mutation
<160>16
<210>1
<211>24
<212>DNA
＜213〉artificial sequence
<220>
＜223〉the ADH1B upstream primer of chemosynthesis
<400>1
agagtgttgg?agaaggggtg?acta
<210>2
<211>24
<212>DNA
＜213〉artificial sequence
<220>
＜223〉the ADH1B downstream primer of chemosynthesis
<400>2
aaatgtattt?gagggttttg?ctac
<210>3
<211>24
<212>DNA
＜213〉artificial sequence
<220>
＜223〉the ADH1C upstream primer of chemosynthesis
<400>3
aaagtttaga?aaaagtgctg?catt
<210>4
<211>24
<212>DNA
＜213〉artificial sequence
<220>
＜223〉the ADH1C downstream primer of chemosynthesis
<400>4
tagcagagaa?tgaaaaggtg?gaag
<210>5
<211>24
<212>DNA
＜213〉artificial sequence
<220>
＜223〉the ALDH2 upstream primer of chemosynthesis
<400>5
ctgctggtgg?ctcggagcct?gctg
<210>6
<211>24
<212>DNA
＜213〉artificial sequence
<220>
＜223〉the ALDH2 downstream primer of chemosynthesis
<400>6
aaagacgtta?gagcaaagtt?taat
<210>7
<211>24
<212>DNA
＜213〉artificial sequence
<220>
＜223〉the CYP2E1 upstream primer of chemosynthesis
<400>7
agtgatttgg?ctggattgta?aatg
<210>8
<211>24
<212>DNA
＜213〉artificial sequence
<220>
＜223〉the CYP2E1 downstream primer of chemosynthesis
<400>8
ctgccgcctc?ctcctctctt?tcct
<210>9
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223〉the ADH1B TaqMan-MGB probe of chemosynthesis
<400>9
gtcatctgtg?tgacagattc?c
<210>10
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223〉the ADH1B TaqMan-MGB probe of chemosynthesis
<400>10
gtcatctgtg?cgacagattc?c
<210>11
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223〉the ADH1C TaqMan-MGB probe of chemosynthesis
<400>11
aaaaggtaaa?acatttgtta?t
<210>12
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223〉the ADH1C TaqMan-MGB probe of chemosynthesis
<400>12
aaaaggtaaa?atatttgtta?t
<210>13
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223〉the ALDH2TaqMan-MGB probe of chemosynthesis
<400>13
gttttcactt?tagtgtatgc?c
<210>14
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223〉the ALDH2TaqMan-MGB probe of chemosynthesis
<400>14
gttttcactt?cagtgtatgc?c
<210>15
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223〉the CYP2E1TaqMan-MGB probe of chemosynthesis
<400>15
ttcaggagag?gtgcagtgtt?a
<210>16
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223〉the CYP2E1TaqMan-MGB probe of chemosynthesis
<400>16
ttcaggagag?ctgcagtgtt?a
Claims (11)
1. be used to detect the primer of ADH1B sudden change, right by the primer that SEQ ID NO:1 in the sequence table and SEQ ID NO:2 form.
2. be used to detect the primer of ADH1C sudden change, right by the primer that SEQ ID NO:3 and SEQ ID NO:4 form.
3. be used to detect the primer of ALDH2 sudden change, right by the primer that SEQ ID NO:5 in the sequence table and SEQ ID NO:6 form.
4. be used to detect the primer of CYP2E1 sudden change, right by the primer that SEQ ID NO:7 in the sequence table and SEQ IDNO:8 form.
5. being used to detect the ADH1B TaqMan-MGB probe of sudden change, is the dna sequence dna of SEQ ID NO:9 and SEQID NO:10 in the sequence table.
6. being used to detect the ADH1C TaqMan-MGB probe of sudden change, is the dna sequence dna of SEQ ID NO:11 and SEQ ID NO:12 in the sequence table.
7. being used to detect the ALDH2 TaqMan-MGB probe of sudden change, is the dna sequence dna of SEQ ID NO:13 and SEQ ID NO:14 in the sequence table.
8. being used to detect the CYP2E1TaqMan-MGB probe of sudden change, is the dna sequence dna of SEQ ID NO:15 and SEQ ID NO:16.
9. according to the described dna sequence dna of claim 5-8, it is characterized in that: 5 ' end is marked with the different report fluorophor of glow color, and 3 ' end not only is marked with non-luminous cancellation group, also is connected with dihydro ring annulated indole porphyrin-tripeptides.
10. according to the described TaqMan-MGB probe of claim 5-8, it is characterized in that: described report fluorophor is FAM or VIC, and non-luminous quenching group is NFQ.
11. a test kit that detects the alcohol metabolism associated gene mutation comprises the described TaqMan probe of described primer of claim 1-4 and claim 5-8.
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| CN104878119A (en) * | 2015-06-26 | 2015-09-02 | 上海美吉生物医药科技有限公司 | Human ALDH 2 gene detection kit |
| CN105803077A (en) * | 2016-04-22 | 2016-07-27 | 浙江中迪生物科技有限公司 | SNP (single nucleotide polymorphism) detection kit for nitroglycerin drug-sensitive type relevant gene ALDH2 and use method thereof |
| CN106987623A (en) * | 2017-03-20 | 2017-07-28 | 杭州迪安医学检验中心有限公司 | A kind of primer of pyrosequencing joint PCR sequencing PCR detection alcohol metabolism gene and its application |
| CN107058549A (en) * | 2017-05-02 | 2017-08-18 | 天津市康婷生物工程有限公司 | A kind of gene detecting kit assessed for capacity for liquor |
| CN108441553A (en) * | 2018-03-13 | 2018-08-24 | 普迈德（北京）科技有限公司 | It is a kind of it is accurate detection ALDH2 gene pleiomorphisms kit and its application |
| CN110747278A (en) * | 2019-10-29 | 2020-02-04 | 圣湘生物科技股份有限公司 | Composition, kit and method for detecting human ethanol metabolism gene polymorphism |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104878119A (en) * | 2015-06-26 | 2015-09-02 | 上海美吉生物医药科技有限公司 | Human ALDH 2 gene detection kit |
| CN105803077A (en) * | 2016-04-22 | 2016-07-27 | 浙江中迪生物科技有限公司 | SNP (single nucleotide polymorphism) detection kit for nitroglycerin drug-sensitive type relevant gene ALDH2 and use method thereof |
| CN106987623A (en) * | 2017-03-20 | 2017-07-28 | 杭州迪安医学检验中心有限公司 | A kind of primer of pyrosequencing joint PCR sequencing PCR detection alcohol metabolism gene and its application |
| CN106987623B (en) * | 2017-03-20 | 2021-01-26 | 杭州迪安医学检验中心有限公司 | Primer for detecting alcohol metabolism gene by pyrosequencing combined sequencing method and application thereof |
| CN107058549A (en) * | 2017-05-02 | 2017-08-18 | 天津市康婷生物工程有限公司 | A kind of gene detecting kit assessed for capacity for liquor |
| CN107058549B (en) * | 2017-05-02 | 2020-11-20 | 天津市康婷生物工程集团有限公司 | Gene detection kit for alcohol capacity assessment |
| CN108441553A (en) * | 2018-03-13 | 2018-08-24 | 普迈德（北京）科技有限公司 | It is a kind of it is accurate detection ALDH2 gene pleiomorphisms kit and its application |
| CN110747278A (en) * | 2019-10-29 | 2020-02-04 | 圣湘生物科技股份有限公司 | Composition, kit and method for detecting human ethanol metabolism gene polymorphism |
| CN110747278B (en) * | 2019-10-29 | 2023-02-21 | 圣湘生物科技股份有限公司 | Composition, kit and method for detecting human ethanol metabolism gene polymorphism |
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