CN103525904A - Method for detecting genetic types of mitochondria acetaldehyde dehydrogenase by utilizing high-resolution melting curve analysis technology - Google Patents
Method for detecting genetic types of mitochondria acetaldehyde dehydrogenase by utilizing high-resolution melting curve analysis technology Download PDFInfo
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Abstract
The invention discloses a method for detecting genetic types of mitochondria acetaldehyde dehydrogenase by utilizing a high-resolution melting curve analysis technology. The method is characterized by comprising the following steps: adopting a silica gel adsorption method to extract genome DNA (Deoxyribose Nucleic Acid) of an oral epithelial cell/peripheral blood cell sample of a detected person; designing a detection primer containing a target site; carrying out site typing on gene sequences subjected to PCR (Polymerase Chain Reaction) amplification according to the high-resolution melting curve analysis technology. The method has the advantages of high sensitivity, good specificity and high detection speed and can be used for evaluating the effectiveness of treating individual angina by nitroglycerin medicines.
Description
Technical field
The detection method that the present invention relates to a kind of plastosome acetaldehyde dehydrogenase gene somatotype, belongs to technical field of molecular biology.
Background technology
Sublingual administration pannonit is the standard treatments of processing acute angina pectoris.Its pharmacological mechanism is that pannonit produces 1 after metabolism, 2-dinitric acid glycerine, discharge the material nitrogen protoxide of expansion vasoactive simultaneously, nitrogen protoxide is by increasing the content of cyclic guanosine monophosphate (cGMP) in the cell vascular smooth muscle that relaxes, alleviate myocardial ischemia, before and after reducing heart, load, thus allevating angina pectoris symptom.
Plastosome acetaldehyde dehydrogenase (ALDH2) is positioned 12q24.2, total length 43.4kb, and containing 13 exons, 518 amino acid of encoding.Its major function is, in liver, the further dehydrogenation of alcohol metabolism product acetaldehyde is become to acetic acid, enters the tricarboxylic acid cycle of peripheral tissues, decomposes and produces CO2 and water.Recently research is found, ALDH2 plays an important role in the biotransformation of pannonit, pannonit is activated and discharge nitric oxide production key enzyme, there is esterase activity, can produce 1 by specific metabolic pannonit, 2-dinitric acid glycerine, nitrite and nitrogen protoxide, realize vasodilation.The vasodilation that studies show that pannonit mediation is that cGMP relies on, and all can be blocked by ALDH2 inhibitor in vivo and in vitro, but not the vasodilation that cGMP relies on is not affected; In being subject to the tissue that pannonit suppresses, the enzymic activity of the ALDH2 even complete deactivation that obviously declines, prompting nitrate tolerance may be relevant with plastosome acetaldehyde dehydrogenase afunction.
There is a G/A mononucleotide polymorphic in ALDH2 the 12nd exon, directly causes the 504th L-glutamic acid to be substituted (Glu504Lys) by Methionin, and the albumen of sudden change has been lost enzymic activity substantially.The gene frequency of this mononucleotide polymorphic in comprising the asian population of Chinese population is the highest, approximately 30%, coded albumen has normal enzymic activity to the homozygote of allelotrope G (being labeled as ADLH2*1/ALDH2* 1), and the coded protease activity sexual abnormality of homozygote (being labeled as ALDH2*2/ALDH2*2) of heterozygote (being labeled as ALDH2*1/ALDH2*2) and allelotrope A.Find 80 people in the association analysis research of sublingual administration pannonit treatment stenocardia clinical efficacy and individual plastosome acetaldehyde dehydrogenase gene type in, there are 59 people to pannonit effective (73.7%), wherein the homozygous individuality of allelotrope G is efficient (has 40 people in 47 people, 85.1%) than the individuality that at least carries an allelotrope A is efficient, (in 33 people, there are 19 people, 57.6%) height, than number ratio, be 4.21(99% credibility interval, 1.51 ~ 11.7).Result shows sublingual administration pannonit treatment stenocardia and plastosome acetaldehyde dehydrogenase gene type significant correlation (χ
2=7.59,
p=0.006), through significant correlation still after the factor correction such as sex, age and disease severity.And heterozygote and homozygote saltant type ALDH2 are compared to wild-type ALDH2 in pannonit drug metabolism processes, Km value raises, Vmax value significantly reduces, the catalytic efficiency of ALDH2 Glu504 enzyme exceeds 10 times than Lys504, point out the polymorphic enzymic activity that can affect ALDH2 of this Lys504, reduce the bio-transformation ability of pannonit.As can be seen here, plastosome acetaldehyde dehydrogenase Glu504Lys can affect the bioactive conversion of pannonit, by the genotype in individual ALDH2 gene Glu504Lys site is detected, can be for assessment of pannonit class medicine to treating individual anginal validity.
Generally the genotype tests method of application is Taqman probe method and DNA direct sequencing at present.Taqman fluorescent probe two ends are mark reporter group and quenching group respectively, and probe be not combined with target sequence while keeping complete, and it is separated that fluorescence report group and quenching group do not have, and fluorescent signal is quenched group and absorbs, and can not be excited to send fluorescence; During pcr amplification, 5 '-3 ' 5 prime excision enzyme activity being had by Taq enzyme after complete complementary pairing is cut degraded by probe enzyme, fluorescence report group is separated with quenching group, and fluorescence detecting system can be received fluorescent signal, and accumulation and the PCR product of realizing fluorescent signal form Complete Synchronization.If there is base mismatch in probe and target sequence, will greatly reduce tightness that probe is combined with target sequence and the activity of Taq enzyme 5 ' end excision enzyme, affect the fluorescence burst size of probe, make to be identified with the DNA sequence dna of different bases.This method steps is few, is difficult for polluting, and is convenient to large sample to carry out somatotype; But design of primers is had to certain restriction, near sequence SNP, there are certain requirements, limited to by mutating alkali yl site and type.DNA direct sequencing can directly provide base information accurately, is treated as the gold standard that sudden change detects, but exist, wastes time and energy, and cost is more expensive, is not suitable for shortcomings such as great amount of samples detect.
The present invention applies a kind of brand-new sudden change scanning and the genetic analysis method of gene type---high-resolution fusion curve (High resolution melting, HRM) analytical technology.Its principle is length, GC content and the base complement difference according to DNA sequence dna, the melting curve of application of high resolution is analyzed sample, saturability fluorescence dye high density occupies all base pairs of double-stranded DNA, when unwind in double-stranded DNA part, fluorescence dye discharges, and the reduction of fluorescence intensity precisely reflects the situation of unwinding of DNA molecular reliably.HRM is not limited to by mutating alkali yl site and type, without sequence-specific probe, adopts novel saturable dye to be easier to detect single base mutation, small segment insertion or disappearance.The method is compared with other genetic typing technology, simple to operate, has the advantages such as highly sensitive, specificity good, cost is low, quick, high throughput testing, and result is accurate, and has realized real stopped pipe operation.
Summary of the invention
The detection method that the object of this invention is to provide a kind of simple plastosome acetaldehyde dehydrogenase gene somatotype.
In order to achieve the above object, the invention provides a kind of method of utilizing high-resolution fusion curve analytical technology detection line plastochondria acetaldehyde dehydrogenase gene somatotype, it is characterized in that, concrete steps are:
The first step: adopt the genomic dna of silica gel adsorption extracting measured mouth epithelial cells/peripheral blood cells sample, adopt running gel imaging method to detect the concentration of DNA and purity, sample to be tested concentration markization is to 10ng/ul; The negative contrast of sample DNA that known ALDH2 Glu504Lys loci gene type is GG, the positive contrast 1 of sample DNA that genotype is AA, the positive contrast 2 that genotype is AG.
Second step: the ALDH2 gene reverse primer solution that the ALDH2 gene forward primer solution that is 10umol/L with sterilized water compound concentration and concentration are 10umol/L; The sequence of ALDH2 gene forward primer is: 5 '-GATGTGTTTGGAGCCCAGTC-3 ', the sequence of ALDH2 gene reverse primer is: 5 '-CAGGTCCCACACTCACAGTTT-3 '.
The 3rd step: the ALDH2 gene forward primer solution 0.5ul, the ALDH2 gene reverse primer solution 0.5ul that add successively Type-it HRM PCR Mix 7.5ul and second step gained in each PCR reacting hole, then detection sample, negative control product and each 2ul of positive reference substance of in different PCR reacting holes, adding respectively the first step to obtain, supply 15ul with sterilizing double distilled water; On Rotor-Gene Q, react, PCR reaction conditions is 92-97 ℃ of sex change 5-15 minute, and 92-97 ℃ of sex change 10-30 second, 57-65 ℃ of annealing 10-30 second, 70-75 ℃ is extended 10-30 second, 30-50 circulation; HRM reaction conditions: 92-97 ℃ sex change 1 minute, 40 ℃ of renaturation 1 minute, initial melting temperature (Tm) 60-65 ℃ of start program heats up and melts to 95 ℃, Real-Time Monitoring fluorescent signal in process, 30-50 time is per second.
The 4th step: application Rotor-Gene Q software analysis HRM result, represents different genotype by typical curve based on curve offset (homozygote) and curve shape variation (heterozygote).After definition known sample genotype, software can recall the genotype of all detection samples automatically.
The present invention detects the genotype in ALDH2 gene Glu504Lys site, can be used for assessing pannonit class medicine to treating individual anginal validity.
The present invention is based on HRM analytical technology, without sequence-specific probe, adopt novel saturable dye, simple to operate, not only there is highly sensitive, the advantage such as specificity good, cost is low, detection speed is fast, high-throughput, and resolving power is high, total overall reaction completes in the reaction tubes of sealing, has effectively avoided crossed contamination.
Accompanying drawing explanation
Fig. 1 is the agarose gel electrophoresis figure of embodiment of the present invention pcr amplification product;
Fig. 2 is the sequencer map of embodiment of the present invention sample negative control;
Fig. 3 is the sequencer map of embodiment of the present invention sample positive control 1;
Fig. 4 is the sequencer map of embodiment of the present invention sample positive control 2;
Fig. 5 is the HRM canonical plotting of embodiment of the present invention large sample;
Fig. 6 is the HRM difference curve figure of embodiment of the present invention large sample.
Embodiment
Below in conjunction with drawings and Examples, the invention will be further described.
Embodiment
1, adopt silica gel adsorption extracting measured mouth epithelial cells genomic dna, running gel imaging method detects the concentration of DNA and purity, and sample to be tested concentration markization is to 10ng/ul;
It is qualified that negative control DNA is considered as while meeting following condition: A260/A280 is between 1.8-2.0 for OD value; Electrophoretic band is clear; Order-checking identifies that ALDH2 Glu504Lys loci gene type is GG, and sequencing result as shown in Figure 2;
It is qualified that positive control 1 DNA is considered as while meeting following condition: A260/A280 is between 1.8-2.0 for OD value; Electrophoretic band is clear; Order-checking identifies that ALDH2 Glu504Lys loci gene type is AA, and sequencing result as shown in Figure 3;
It is qualified that positive control 2 DNA are considered as while meeting following condition: A260/A280 is between 1.8-2.0 for OD value; Electrophoretic band is clear; Order-checking identifies that ALDH2 Glu504Lys loci gene type is AG, and sequencing result as shown in Figure 4.
2, according to the conservative region of ALDH2 gene, adopt online software Primer 3 design primers, determine that best primer is 18-24bp size, PCR product length 70-150bp, target site peripheral position is avoided other SNP sites (limited-liability company is synthetic by life work biotechnology (Shanghai)).The ALDH2 gene reverse primer solution that the ALDH2 gene forward primer solution that is 10umol/L with sterilized water compound concentration and concentration are 10umol/L; The sequence of ALDH2 gene forward primer is: 5 '-GATGTGTTTGGAGCCCAGTC-3 ', the sequence of ALDH2 gene reverse primer is: 5 '-CAGGTCCCACACTCACAGTTT-3 '.
3, in each PCR reacting hole, add successively Type-it HRM PCRMix 7.5ul(QIAGEN company to produce, comprise 2 x HRM PCR Master Mix, 10 * PCR damping fluid, Q-solution, EvaGreen Dye, HotStarTaq DNA polysaccharase) and ALDH2 gene forward primer solution 0.5ul, the ALDH2 gene reverse primer solution 0.5ul of step 2 gained, then detection sample, negative control product and each 2ul of positive reference substance of in different PCR reacting holes, adding respectively step 1 to obtain, supply 15ul with sterilizing double distilled water; On Rotor-Gene Q, react, PCR reaction conditions is 95 ℃ of sex change 10 minutes, 95 ℃ of sex change 10 seconds, and 57 ℃ of annealing 10 seconds, 72 ℃ are extended 10 seconds, 40 circulations; HRM reaction conditions: 95 ℃ of sex change 1 minute, 40 ℃ of renaturation 1 minute, 65 ℃ of start programs of initial melting temperature (Tm) heat up and melt to 95 ℃, Real-Time Monitoring fluorescent signal in process, 40 times are per second.
4, application Rotor-Gene Q software analysis HRM result, as shown in Figure 5, in the DNA detection of extracting at 10 routine oral mucosa epithelial cells, having 6 routine ALDH2 Glu504Lys loci gene types is GG, and 3 routine genotype are AG, and 1 routine genotype is AA.
<110> Shanghai Zhongyou Medicine High-tech Co., Ltd.
<120>utilize the method for high-resolution fusion curve analytical technology detection line plastochondria acetaldehyde dehydrogenase gene somatotype
<160>?3
<210>?1
<211>?300
<212>?DNA
<213>people (Homo sapiens)
<220>
<221>?mutation
<222>?(101)
<223>n=a or g
<400>?1
caactgctat?gatgtgtttg?gagcccagtc?accctttggt?ggctacaaga?tgtcggggag?60
tggccgggag?ttgggcgagt?acgggctgca?ggcatacact?naagtgaaaa?ctgtgagtgt?120
gggacctgct?gggggctcag?ggcctgttgg?ggcttgaggg?tctgctggtg?gctcggagcc?180
tgctggggga?ttggggtctg?ttgggggctc?ggggcctgcc?agaggttcag?gacctgccgg?240
ggactcaggg?cctgctggaa?gttcaggacc?tgctggggat?cagggcctgc?cagggatttag?300
<210>2
<211>20
<212>DNA
<213>artificial sequence
<220>
<223>for the forward primer of ALDH2 Glu504Lys SNP sequence amplification.
<400>2
gatgtgtttg?gagcccagtc?20
<210>3
<211>21
<212>DNA
<213>artificial sequence
<220>
<223>for the reverse primer of ALDH2 Glu504Lys SNP sequence amplification.
<400>3
caggtcccac?actcacagttt?21
Claims (1)
1. in a kind of method of utilizing high-resolution fusion curve analytical technology detection line plastochondria acetaldehyde dehydrogenase gene somatotype, it is characterized in that, concrete steps are:
The first step: adopt the genomic dna of silica gel adsorption extracting measured mouth epithelial cells/peripheral blood cells sample, running gel imaging method detects the concentration of DNA and purity, and sample to be tested concentration markization is to 10ng/ul; The negative contrast of sample DNA that known ALDH2 Glu504Lys loci gene type is GG, the positive contrast 1 of sample DNA that genotype is AA, the positive contrast 2 that genotype is AG;
Second step: the ALDH2 gene reverse primer solution that the ALDH2 gene forward primer solution that is 10umol/L with sterilized water compound concentration and concentration are 10umol/L; The sequence of ALDH2 gene forward primer is: 5 '-GATGTGTTTGGAGCCCAGTC-3 ', the sequence of ALDH2 gene reverse primer is: 5 '-CAGGTCCCACACTCACAGTTT-3 ';
The 3rd step: the ALDH2 gene forward primer solution 0.5ul, the ALDH2 gene reverse primer solution 0.5ul that add successively Type-it HRM PCR Mix 7.5ul and second step gained in each PCR reacting hole, then detection sample, negative control product and each 2ul of positive reference substance of in different PCR reacting holes, adding respectively the first step to obtain, supply 15ul with sterilizing double distilled water; On Rotor-Gene Q, react, PCR reaction conditions is 92-97 ℃ of sex change 5-15 minute, and 92-97 ℃ of sex change 10-30 second, 57-65 ℃ of annealing 10-30 second, 70-75 ℃ is extended 10-30 second, 30-50 circulation; HRM reaction conditions: 92-97 ℃ sex change 1 minute, 40 ℃ of renaturation 1 minute, initial melting temperature (Tm) 60-65 ℃ of start program heats up and melts to 95 ℃, Real-Time Monitoring fluorescent signal in process, 30-50 time is per second;
The 4th step: application Rotor-Gene Q software analysis HRM result, represents different genotype by typical curve based on curve offset (homozygote) and curve shape variation (heterozygote); After definition known sample genotype, software can recall the genotype of all detection samples automatically.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103834733A (en) * | 2014-02-27 | 2014-06-04 | 厦门大学附属中山医院 | Kit and method for determining mutation sites of genes of acetaldehyde dehydrogenase 2 and methylene tetrahydrofolic acid reductase by virtue of single tube at the same time |
| CN105400871A (en) * | 2015-11-16 | 2016-03-16 | 北京晋祺生物科技有限公司 | Detection primer group for ALDH2 genes, reaction system comprising same and application |
| CN107267650A (en) * | 2017-08-11 | 2017-10-20 | 踏石生物科技（苏州）有限公司 | A kind of ALDH2*2 genotype detections kit and its detection method |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102876782A (en) * | 2012-09-13 | 2013-01-16 | 周宏灏 | Kit for detecting acetaldehyde dehydrogenase 2 (ALDH2) gene polymorphism by pyro-sequencing method and method |
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2013
- 2013-07-11 CN CN201310288926.0A patent/CN103525904A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102876782A (en) * | 2012-09-13 | 2013-01-16 | 周宏灏 | Kit for detecting acetaldehyde dehydrogenase 2 (ALDH2) gene polymorphism by pyro-sequencing method and method |
Non-Patent Citations (1)
| Title |
|---|
| 袁晓文: "高分辨率熔解曲线分析技术用于乙醇脱氢酶1B和乙醛脱氢酶2基因的快速分型", 《中国优秀硕士学位论文数据库》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103834733A (en) * | 2014-02-27 | 2014-06-04 | 厦门大学附属中山医院 | Kit and method for determining mutation sites of genes of acetaldehyde dehydrogenase 2 and methylene tetrahydrofolic acid reductase by virtue of single tube at the same time |
| CN105400871A (en) * | 2015-11-16 | 2016-03-16 | 北京晋祺生物科技有限公司 | Detection primer group for ALDH2 genes, reaction system comprising same and application |
| CN107267650A (en) * | 2017-08-11 | 2017-10-20 | 踏石生物科技（苏州）有限公司 | A kind of ALDH2*2 genotype detections kit and its detection method |
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Application publication date: 20140122 |