CN1268769C - Polymorphism detection chip for gene of enzyme relevant to ethanol metabolism, preparation method of usage - Google Patents
Polymorphism detection chip for gene of enzyme relevant to ethanol metabolism, preparation method of usage Download PDFInfo
- Publication number
- CN1268769C CN1268769C CN 200410017946 CN200410017946A CN1268769C CN 1268769 C CN1268769 C CN 1268769C CN 200410017946 CN200410017946 CN 200410017946 CN 200410017946 A CN200410017946 A CN 200410017946A CN 1268769 C CN1268769 C CN 1268769C
- Authority
- CN
- China
- Prior art keywords
- gta
- aag
- ttg
- cat
- ccg
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 33
- 108090000623 proteins and genes Proteins 0.000 title abstract description 20
- 238000002360 preparation method Methods 0.000 title abstract description 11
- 102000004190 Enzymes Human genes 0.000 title abstract description 6
- 238000001514 detection method Methods 0.000 title abstract description 4
- 230000004149 ethanol metabolism Effects 0.000 title 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 48
- 102100034044 All-trans-retinol dehydrogenase [NAD(+)] ADH1B Human genes 0.000 claims abstract description 33
- 101710193111 All-trans-retinol dehydrogenase [NAD(+)] ADH4 Proteins 0.000 claims abstract description 25
- 102100034042 Alcohol dehydrogenase 1C Human genes 0.000 claims abstract description 24
- 239000000523 sample Substances 0.000 claims abstract description 24
- 101000796894 Coturnix japonica Alcohol dehydrogenase 1 Proteins 0.000 claims abstract description 16
- 101000780463 Homo sapiens Alcohol dehydrogenase 1C Proteins 0.000 claims abstract description 16
- 108010009513 Mitochondrial Aldehyde Dehydrogenase Proteins 0.000 claims abstract description 16
- 102000009645 Mitochondrial Aldehyde Dehydrogenase Human genes 0.000 claims abstract description 15
- SNBCLPGEMZEWLU-QXFUBDJGSA-N 2-chloro-n-[[(2r,3s,5r)-3-hydroxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methyl]acetamide Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CNC(=O)CCl)[C@@H](O)C1 SNBCLPGEMZEWLU-QXFUBDJGSA-N 0.000 claims abstract description 13
- 230000004060 metabolic process Effects 0.000 claims description 29
- 238000012360 testing method Methods 0.000 claims description 13
- 108700028369 Alleles Proteins 0.000 abstract description 5
- 230000008901 benefit Effects 0.000 abstract description 3
- 230000002503 metabolic effect Effects 0.000 abstract description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N acetic acid Substances CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 14
- 101150016330 ADH3 gene Proteins 0.000 description 10
- 238000013461 design Methods 0.000 description 10
- 101150078509 ADH2 gene Proteins 0.000 description 9
- 101150038502 ALDH2 gene Proteins 0.000 description 9
- 238000005406 washing Methods 0.000 description 9
- 208000022309 Alcoholic Liver disease Diseases 0.000 description 8
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 8
- 150000001299 aldehydes Chemical class 0.000 description 8
- 239000002751 oligonucleotide probe Substances 0.000 description 8
- 210000004185 liver Anatomy 0.000 description 7
- 101100441878 Caenorhabditis elegans cyn-3 gene Proteins 0.000 description 6
- 101100464856 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) cyp-3 gene Proteins 0.000 description 6
- 108091034117 Oligonucleotide Proteins 0.000 description 6
- 108010044467 Isoenzymes Proteins 0.000 description 5
- 206010067125 Liver injury Diseases 0.000 description 5
- 230000001476 alcoholic effect Effects 0.000 description 5
- 231100000234 hepatic damage Toxicity 0.000 description 5
- 230000008818 liver damage Effects 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 230000011664 signaling Effects 0.000 description 5
- SJECZPVISLOESU-UHFFFAOYSA-N 3-trimethoxysilylpropan-1-amine Chemical compound CO[Si](OC)(OC)CCCN SJECZPVISLOESU-UHFFFAOYSA-N 0.000 description 4
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 4
- 229960000583 acetic acid Drugs 0.000 description 4
- 235000011114 ammonium hydroxide Nutrition 0.000 description 4
- KRVSOGSZCMJSLX-UHFFFAOYSA-L chromic acid Substances O[Cr](O)(=O)=O KRVSOGSZCMJSLX-UHFFFAOYSA-L 0.000 description 4
- 238000010511 deprotection reaction Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000005357 flat glass Substances 0.000 description 4
- AWJWCTOOIBYHON-UHFFFAOYSA-N furo[3,4-b]pyrazine-5,7-dione Chemical compound C1=CN=C2C(=O)OC(=O)C2=N1 AWJWCTOOIBYHON-UHFFFAOYSA-N 0.000 description 4
- 239000012362 glacial acetic acid Substances 0.000 description 4
- 238000009396 hybridization Methods 0.000 description 4
- 239000006210 lotion Substances 0.000 description 4
- 239000002773 nucleotide Substances 0.000 description 4
- 125000003729 nucleotide group Chemical group 0.000 description 4
- 238000004321 preservation Methods 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 238000004506 ultrasonic cleaning Methods 0.000 description 4
- 101150028074 2 gene Proteins 0.000 description 3
- 108010021809 Alcohol dehydrogenase Proteins 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000014509 gene expression Effects 0.000 description 3
- IKHGUXGNUITLKF-UHFFFAOYSA-N Acetaldehyde Chemical compound CC=O IKHGUXGNUITLKF-UHFFFAOYSA-N 0.000 description 2
- 208000007848 Alcoholism Diseases 0.000 description 2
- 108020002663 Aldehyde Dehydrogenase Proteins 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 2
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- FQVLRGLGWNWPSS-BXBUPLCLSA-N (4r,7s,10s,13s,16r)-16-acetamido-13-(1h-imidazol-5-ylmethyl)-10-methyl-6,9,12,15-tetraoxo-7-propan-2-yl-1,2-dithia-5,8,11,14-tetrazacycloheptadecane-4-carboxamide Chemical compound N1C(=O)[C@@H](NC(C)=O)CSSC[C@@H](C(N)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](C)NC(=O)[C@@H]1CC1=CN=CN1 FQVLRGLGWNWPSS-BXBUPLCLSA-N 0.000 description 1
- 101150084750 1 gene Proteins 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 102000007698 Alcohol dehydrogenase Human genes 0.000 description 1
- 102100034035 Alcohol dehydrogenase 1A Human genes 0.000 description 1
- 102000005369 Aldehyde Dehydrogenase Human genes 0.000 description 1
- 208000019901 Anxiety disease Diseases 0.000 description 1
- 208000008964 Chemical and Drug Induced Liver Injury Diseases 0.000 description 1
- 238000000018 DNA microarray Methods 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- 206010072268 Drug-induced liver injury Diseases 0.000 description 1
- 208000004930 Fatty Liver Diseases 0.000 description 1
- 101000892220 Geobacillus thermodenitrificans (strain NG80-2) Long-chain-alcohol dehydrogenase 1 Proteins 0.000 description 1
- 206010019708 Hepatic steatosis Diseases 0.000 description 1
- 208000005176 Hepatitis C Diseases 0.000 description 1
- 206010019728 Hepatitis alcoholic Diseases 0.000 description 1
- 206010019799 Hepatitis viral Diseases 0.000 description 1
- 101000780443 Homo sapiens Alcohol dehydrogenase 1A Proteins 0.000 description 1
- XUYPXLNMDZIRQH-LURJTMIESA-N N-acetyl-L-methionine Chemical compound CSCC[C@@H](C(O)=O)NC(C)=O XUYPXLNMDZIRQH-LURJTMIESA-N 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 206010001584 alcohol abuse Diseases 0.000 description 1
- 201000007930 alcohol dependence Diseases 0.000 description 1
- 208000025746 alcohol use disease Diseases 0.000 description 1
- 208000002353 alcoholic hepatitis Diseases 0.000 description 1
- 108010081577 aldehyde dehydrogenase (NAD(P)+) Proteins 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 230000036506 anxiety Effects 0.000 description 1
- 230000037429 base substitution Effects 0.000 description 1
- 208000019425 cirrhosis of liver Diseases 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 230000001351 cycling effect Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 208000022602 disease susceptibility Diseases 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000035622 drinking Effects 0.000 description 1
- 231100000594 drug induced liver disease Toxicity 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
- 208000010706 fatty liver disease Diseases 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 238000012252 genetic analysis Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 229960002989 glutamic acid Drugs 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 208000002672 hepatitis B Diseases 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 206010025482 malaise Diseases 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 208000030159 metabolic disease Diseases 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 238000005065 mining Methods 0.000 description 1
- 238000013188 needle biopsy Methods 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 230000007096 poisonous effect Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000007894 restriction fragment length polymorphism technique Methods 0.000 description 1
- 239000003620 semiochemical Substances 0.000 description 1
- 238000003201 single nucleotide polymorphism genotyping Methods 0.000 description 1
- 239000012279 sodium borohydride Substances 0.000 description 1
- 229910000033 sodium borohydride Inorganic materials 0.000 description 1
- 231100000240 steatosis hepatitis Toxicity 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 201000001862 viral hepatitis Diseases 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention discloses a chip for detecting the polymorphism of relevant genes of ethanol metabolic enzymes, a preparation method thereof and functions thereof. A carrier has the following probes: ADH3: 5'-AAG GTA AAA CAT TTG TTAT5'-AAG GTA AAA TAT TTG TTAT5'-AAG GTA AAA AAT TTG TTAT5'-AAG GTA AAA GAT TTG TTATA DH2(an allele 1 and an allele 2): 5'-AAT CTG TCG CAC AGA TG5'-AAG GTA AAA CAT TTG TTAT5'-AAT CTG TCC CAC AGA TG5'-AAT CTG TCT CAC AGA TGADH2(the allele 1 and an allele 3): 5'-GCA GTA TCT GTA CCG TC5'-GCA GTA TCC GTA CCG TC5'-GCA GTA TCA GTA CCG TC5'-GCA GTA TCG GTA CCG TC ALDH2: 5'-CAT ACA CTG AAG TGA AA5'-CAT ACA CTA AAG TGA AA5'-CAT ACA CTT AAG TGA AA5'-CAT ACA CTC AAG TGA AA. The present invention has the advantages that the rapid, simple and high-flux detection of the polymorphism of relevant genes of ethanol metabolic enzymes, namely ADH2, ADH3 and ALDH2, is realized, and the chip has significant social and economic benefit.
Description
Technical field
The present invention relates to a kind of alcohol metabolism relative enzyme gene polymorphic test chip.
Background technology
Alcohol abuse and alcohol dependence have become drink more worth people of public health problem serious day by day in the world today, particularly teenager and have paid attention to.The alcoholic liver disease past is more rare in China, also be few to its research therefore, but the sickness rate of alcoholic liver disease obviously increases in recent years, has become the second largest hepatopathy that is only second to viral hepatitis.Alcohol has tangible toxic action to liver, and fatty liver is to a certain degree arranged more than 80% among the heavy drinking essence person, and 10%-35% can develop into alcoholic hepatitis, and 10%-20% will develop into liver cirrhosis.
Alcohol (ethanol) metabolic conversion in vivo is acetaldehyde and acetate, be mainly ethanol dehydrogenase (alcoholdehydrogenase, ADH) and acetaldehyde dehydrogenase (aldehyde dehydrogenase, ALDH) effect.ADH has the isozyme ADH1 (α) of 3 kinds of different genes codings, ADH2 (β), ADH3 (γ), wherein there are certain polymorphism in ADH2 and ADH3, ADH2 has three allelotrope sites, ADH2*1, ADH2*2 and ADH2*3, β 1 encodes respectively, 3 three subunits of β 2 and β, the allelic polymorphism of ADH2 be because 47 (G → A) and 369 (C → T) the single base substitution of codon:, the isozyme of β 1 has low Km (0.049mmol/L) and low maximal clearance (V max) (0.23U/min) to alcohol, the isozyme of β 2. relatively has low Km (0.94mmol/L) maximal clearance (8.6U/min), and isozyme β 3 has high Km of alcohol (36mmol/L) and high Vmax (7.9U/min).The activity of ADH3*1 expression product is 2 times of ADH3*2.The ALDH2 genes encoding has the allelotrope of the subunit of active and non-activity to be named as ALDH2*1 and ALDH2*2 respectively, the difference of these isozyme is the base exchange (G → A), produce the exchange of Methionin (non-activity subunit) and L-glutamic acid (active subunit) of 487 codons.The polymorphism of alcohol metabolism enzyme genes involved plays an important role in the morbidity of alcoholic liver disease and other metabolism and drug-induced liver disease.
Being used for SNPs, to do genetic analysis be to refer in particular to detect a large amount of single nucleotide variations that exist of human genome, this relevant with the poisonous substance susceptibility with disease susceptibility [1].SNPs is not only the mark of human race and individual difference, also is the mark of decision different crowd race and individual difference, can become the searching disease related gene, carries out the basis of medical diagnosis on disease, prevention and drug screening.Being used for the ordinary method that SNPs detects has allele specific oligonucleotide (ASO) hybridization, restriction enzyme enzyme process (RFLP), locus specificity primer PCR (PCR-SSP) and directly check order etc.The detection method that develops into new SNPs of biochip technology provides possibility [2].The oligonucleotide chip technology is a kind of technique of gene detection that newly-developed gets up, it can be arranged in a large amount of oligonucleotide probes on the slide regularly, these probes can show that the complementary sequence of extension increasing sequence of the sample DNA of material mark combines with signal, by semiochemicals is detected, results of hybridization is carried out the software processing analysis, thereby obtain intensity of hybridization signal and distribution pattern figure thereof.
Reference:
[1]Khner M K，Beerli P，Yamato J，et al.Usefulness of single nucleatide polymorphismdata for estimating population parameters.Genetics，2000，156：439-447
[2]Hirschlhorn J N，Sklar P，Lindblad-Toh K，et al.SBE-TAGS：an array-based methodfor efficient single-nucleotide polymorphism genotyping.Proc Natl Acad SciUSA，2000，97：12164-12169
[3]Picoult-Newberg L，Idker TE，Pohl M G，et al.Mining SNPs from ESTdatabases.Genome Res，1999，9：167-174.
[4] anxiety is towards the old Li Hua of brightness, Chen Weixing etc. the preparation and the application of pure metabolizing enzyme oligonucleotide gene chip.China's digestion magazine 2003,23 (11); 695-6
Summary of the invention
The purpose of this invention is to provide a kind of alcohol metabolism relative enzyme gene polymorphic test chip.
Alcohol metabolism relative enzyme gene polymorphic test chip: on carrier, be provided with following probe:
ADH3： 5’-AAG GTA AAA CAT TTG TTAT
5’-AAG GTA AAA TAT TTG TTAT
5’-AAG GTA AAA AAT TTG TTAT
5’-AAG GTA AAA GAT TTG TTAT
ADH2 (allelotrope 1 and allelotrope 2): 5 '-AAT CTG TCG CAC AGA TG
5’-AAG GTA AAA CAT TTG TTAT
5’-AAT CTG TCC CAC AGA TG
5’-AAT CTG TCT CAC AGA TG
ADH2 (allelotrope 1 and allelotrope 3): 5 '-GCA GTA TCT GTA CCG TC
5’-GCA GTA TCC GTA CCG TC
5’-GCA GTA TCA GTA CCG TC
5’-GCA GTA TCG GTA CCG TC
ALDH2： 5’-CAT ACA CTG AAG TGA AA
5’-CAT ACA CTA AAG TGA AA
5’-CAT ACA CTT AAG TGAAA
5’-CAT ACA CTC AAG TGAAA
Alcohol metabolism relative enzyme gene polymorphic test chip preparation method step is:
1) processing of chip carrier: wave carrier piece chromic acid lotion soaked overnight, washing, spend the night in 24～26% ammoniacal liquor, washing, sheet glass immerses in 95% ethanolic soln of aminopropyl trimethoxysilane, regulates PH to 4～4.5,95% ethanol ultrasonic cleaning with Glacial acetic acid, dried 3～5 hours for 150～160 ℃, 9～11% glutaraldehyde are processed into aldehyde radicalization;
2) primer and probe is synthetic: at alcohol metabolism enzyme genes involved ADH2, ADH3 and ALDH2 gene design one cover probe (as table 1), 4 pairs of primers (as table 2) have been synthesized at alcohol metabolism enzyme genes involved ADH2, ADH3 and ALDH2 gene design, a Cyp-3 signaling molecule mark is arranged in every pair of primer, synthetic back 75～85 ℃ of deprotections of strong aqua, cut 14～17 hours, the OPC column purification, the quantitative final vacuum of ultraviolet is dry to be concentrated, and-20～-80 ℃ of preservations are standby;
3) gene chip preparation and aftertreatment: oligonucleotide probe is dissolved in 3 * SSC solution with the concentration of 15 μ M, oligonucleotide is sprayed onto on the slide of aldehyde radicalization with PixySys5500 chip preparing instrument, and dot spacing is 0.5mm.After point sample finished, room temperature was placed and is spent the night.
The polymorphism that alcohol metabolism relative enzyme gene polymorphic test chip is used for alcoholic liver patient alcohol metabolism enzyme genes involved ADH2, ADH3 and ALDH2 detects and somatotype.
The present invention will be through the probe point sample selected on slide, hybridize respectively with through alcohol metabolism enzyme genes involved ADH2, the ADH3 of pcr amplification and the dna fragmentation of ALDH2, the polymorphic situation in each site of disposable acquisition, thereby realize alcohol metabolism enzyme related gene polymorphism is carried out quick, simple and direct, high throughput testing and somatotype, have important society and economic benefit.
Embodiment
Immobilised probe is an oligonucleotide probe on the slide of the present invention, its sequence plays length range 15-20 base according to the gene expression characteristics design of alcohol metabolism enzyme genes involved ADH2, ADH3 and ALDH2, is generally 18 bases, its Tm value basically identical is at 40～43 ℃.
Said primer is the gene expression characteristics design according to alcohol metabolism enzyme genes involved ADH2, ADH3 and ALDH2, and primer sequence length is respectively applied for the amplification of said gene between 20-24 base.
The signaling molecule mark is the primer at the gene order amplification of alcohol metabolism enzyme genes involved ADH2, ADH3 and ALDH2.The signaling molecule mark is a fluorescence molecule.
Table 1: the probe of alcohol metabolism relative enzyme gene polymorphic test chip
| gene | probe |
| ADH3 | 5’-AAG GTA AAA CAT TTG TTAT |
| 5’-AAG GTA AAA TAT TTG TTAT | |
| 5’-AAG GTA AAA AAT TTG TTAT | |
| 5’-AAG GTA AAA GAT TTG TTAT | |
| ADH2 (allelotrope 1 and allelotrope 2) | 5’-AAT CTG TCG CAC AGA TG |
| 5’-AAT CTG TCA CAC AGA TG | |
| 5’-AAT CTG TCC CAC AGA TG | |
| 5’-AAT CTG TCT CAC AGA TG | |
| ADH2 (allelotrope 1 and allelotrope 3) | 5’-GCA GTA TCT GTA CCG TC |
| 5’-GCA GTA TCC GTA CCG TC | |
| 5’-GCA GTA TCA GTA CCG TC | |
| 5’-GCA GTA TCG GTA CCG TC | |
| ALDH2 | 5’-CAT ACA CTG AAG TGA AA |
| 5’-CAT ACA CTA AAG TGA AA | |
| 5’-CAT ACA CTT AAG TGA AA | |
| 5’-CAT ACA CTC AAG TGA AA |
Table 2: the primer of alcohol metabolism relative enzyme gene polymorphic test chip
| Gene | The Cyp-3 fluorescent dye primer | Primer |
| ADH3 | 5’-CTT TAA GAG TAA AGA ATC TGT CC-3’ | 5’-ACC TCT TTC CAG AGC GAA GCAG-3’ |
| ADH2 (allelotrope 1 and allelotrope 2) | 5’-GAA GGG GGG TCA CCA GGT TGC-3’ | 5’-AAT CTT TTC TGA ATC TGA ACAG-3’ |
| ADH2 (allelotrope 1 and allelotrope 3) | 5’-TTG ATA ACA TCT CTG AAG AGC TGA-3’ | 5’-TGG ACT CTC ACA ACA AGC ATGT-3’ |
| ALDH2 | 5’-CAG GTC CCA CAC TCA CAG TTT-3’ | 5’-CAC CCT TTG GTG GCT ACA AG-3’ |
Embodiment 1: the preparation of nucleotide gene chip:
1) processing of chip carrier: wave carrier piece chromic acid lotion soaked overnight, washing is spent the night in 25% ammoniacal liquor, washing.Sheet glass immerses in 95% ethanolic soln of aminopropyl trimethoxysilane, regulates PH to 4.5 with Glacial acetic acid, and 95% ethanol ultrasonic cleaning was dried 4 hours for 160 ℃, and 5% glutaraldehyde is processed into aldehyde radicalization.
2) primer and probe is synthetic: the one cover probe (seeing Table 1) that the present invention is directed to alcohol metabolism enzyme genes involved ADH2, ADH3 and ALDH2 gene design.4 pairs of primers (seeing Table 2) have been synthesized at alcohol metabolism enzyme genes involved ADH2, ADH3 and ALDH2 gene design.A Cyp-3 fluorescent mark is arranged in every pair of primer.Synthetic back was cut the OPC column purification 15 hours with 80 ℃ of deprotections of strong aqua.The quantitative final vacuum of ultraviolet is dry to be concentrated, and-20 ℃ of preservations are standby.
3) gene chip preparation and aftertreatment: oligonucleotide probe is dissolved in 3 * SSC solution with the concentration of 15 μ M, oligonucleotide is sprayed onto on the slide of aldehyde radicalization with PixySys5500 chip preparing instrument, and dot spacing is 0.5mm.After point sample finished, room temperature was placed and is spent the night.
Embodiment 2: the preparation of nucleotide gene chip:
1) processing of chip carrier: wave carrier piece chromic acid lotion soaked overnight, washing, spend the night in 24% ammoniacal liquor, washing, sheet glass immerses in 95% ethanolic soln of aminopropyl trimethoxysilane, regulates PH to 4,95% ethanol ultrasonic cleaning with Glacial acetic acid, dried 3 hours for 150 ℃, 9% glutaraldehyde is processed into aldehyde radicalization;
2) primer and probe is synthetic: at alcohol metabolism enzyme genes involved ADH2, ADH3 and ALDH2 gene design one cover probe, 4 pairs of primers have been synthesized at alcohol metabolism enzyme genes involved ADH2, ADH3 and ALDH2 gene design, a Cyp-3 signaling molecule mark is arranged in every pair of primer, synthetic back 75 ℃ of deprotections of strong aqua, cut 13 hours, the OPC column purification, the quantitative final vacuum of ultraviolet is dry to be concentrated, and-20 ℃ of preservations are standby;
3) gene chip preparation and aftertreatment: oligonucleotide probe is dissolved in 3 * SSC solution with the concentration of 15 μ M, oligonucleotide is sprayed onto on the slide of aldehyde radicalization with PixySys5500 chip preparing instrument, and dot spacing is 0.5mm.After point sample finished, room temperature was placed and is spent the night.
The preparation of embodiment 3 nucleotide gene chips:
1) processing of chip carrier: wave carrier piece chromic acid lotion soaked overnight, washing, spend the night in 26% ammoniacal liquor, washing, sheet glass immerses in 95% ethanolic soln of aminopropyl trimethoxysilane, regulates PH to 4.5,95% ethanol ultrasonic cleaning with Glacial acetic acid, dried 5 hours for 160 ℃, 11% glutaraldehyde is processed into aldehyde radicalization;
2) primer and probe is synthetic: at alcohol metabolism enzyme genes involved ADH2, ADH3 and ALDH2 gene design one cover probe, 4 pairs of primers have been synthesized at alcohol metabolism enzyme genes involved ADH2, ADH3 and ALDH2 gene design, a Cyp-3 signaling molecule mark is arranged in every pair of primer, synthetic back 85 ℃ of deprotections of strong aqua, cut 17 hours, the OPC column purification, the quantitative final vacuum of ultraviolet is dry to be concentrated, and-80 ℃ of preservations are standby;
3) gene chip preparation and aftertreatment: oligonucleotide probe is dissolved in 3 * SSC solution with the concentration of 15 μ M, oligonucleotide is sprayed onto on the slide of aldehyde radicalization with PixySys5500 chip preparing instrument, and dot spacing is 0.5mm.After point sample finished, room temperature was placed and is spent the night.
The using method of alcohol metabolism relative enzyme gene polymorphic test chip is:
1) slide that is loaded with oligonucleotide probe uses preceding 0.2%SDS and clear water respectively to wash twice, 1%NaBH4 solution reduction 10min after the dry air, and 0.2%SDS washes once, and washing once with stand-by, is finished probe covalency immobilization in surface of glass slide after the dry air.
2) contain 50ng DNA, 4 pairs of primers (table 2), 10mM Tris-HClpH8.3,50mM KCl, 1.5mM MgCl in the 20ul PCR reaction system
2, 200uM dNTP, and 1.5 Taq of unit enzymes; Wherein primer content is 20ng Cyp-3 fluorescent dye primer, the 2ng general primer.PCR cycling condition: 95 ℃ of sex change in 5 minutes; Each 45 seconds the 94 ℃ of sex change of 35 round-robin, 57.5 ℃ of annealing, 72 ℃ of extensions; Be 72 ℃ of extensions in 5 minutes at last.
3) the 2ulPCR product is with after the 10ul hybridization buffer mixes, hybridized 1 hour at 42 ℃ with the oligonucleotide probe slide for preparing, use 1 * SSC, 0.2%SDS then respectively, 0.2 * SSC, 0.1 * SSC cleans, detect with laser co-focusing fluorescent scanning instrument ScanArray 3000 at last, scanning is 3 times under 85% laser intensity.Analyze positive probe and form, judge the differentiation of the gene pleiomorphism of alcohol metabolism relevant enzyme.Alcohol metabolism relative enzyme gene polymorphic test chip use-case: to 165 routine alcoholists, wherein do not have 43 examples of liver damage, alcoholic liver disease 122 examples, 25～70 years old age, average 44.0 years old.Normal control group 65 examples, 26～68 years old age, average 45.9 years old.All research objects are all from Zhejiang Province's alcoholic liver disease epidemiology survey crowd.Case definition is with reference to " diagnosis basis of alcoholic liver disease and healing, improvement standard [3] ", and gets rid of the hepatitis B surface antigen and the hepatitis C antibody positive, and the person of not being true to type does needle biopsy of liver.All objects all extract the 3ml peripheral blood, extract DNA routinely and detect with the said chip aforesaid method.The result is: the 1) polymorphism of ADH2 gene: the ADH2*1 gene frequency of normal healthy controls group, alcoholist's group, no liver damage group and alcoholic liver disease group is respectively 37.69%, 55.76%, 46.51%, 59.02%; The ADH2*2 gene frequency is respectively 62.31%, 44.24%, 53.49%, 40.98%; Do not find ADH2*3 allelotrope.Alcoholist and alcoholic liver patient's ADH2*1 frequency is apparently higher than the normal healthy controls group, and the alcoholic liver patient is higher than the alcoholist of no liver damage; 2) ADH3 gene pleiomorphism: the gene frequency of the ADH3 gene of alcoholist and alcoholic liver disease and normal healthy controls group compare, and the alcoholist's of no liver damage ADH3*2 frequency is higher than normal healthy controls.3) ALDH2 gene pleiomorphism: alcoholist and alcoholic liver patient's ALDH2*2 gene frequency is lower than the normal healthy controls group, and alcoholic liver patient ALDH2*2 gene frequency significantly is lower than the alcoholist [4] of no liver damage.
Claims (2)
1, a kind of alcohol metabolism relative enzyme gene polymorphic test chip is characterized in that: be provided with following probe on carrier:
ADH3： 5’-AAG GTA AAA CAT TTG TTAT
5’-AAG GTA AAA TAT TTG TTAT
5’-AAG GTA AAA AAT TTG TTAT
5’-AAG GTA AAA GAT TTG TTAT
The allelotrope 1 of ADH2 and allelotrope 2:5 '-AAT CTG TCG CAC AGA TG
5’-AAG GTA AAA CAT TTG TTAT
5’-AAT CTG TCC CAC AGA TG
5’-AAT CTG TCT CAC AGA TG
The allelotrope 1 of ADH2 and allelotrope 3:5 '-GCA GTA TCT GTA CCG TC
5’-GCA GTA TCC GTA CCG TC
5’-GCA GTA TCA GTA CCG TC
5’-GCA GTA TCG GTA CCG TC
ALDH2： 5’-CAT ACA CTG AAG TGA AA
5’-CAT ACA CTA AAG TGA AA
5’-CAT ACA CTT AAG TGA AA
5’-CAT ACA CTC AAG TGA AA。
2, a kind of alcohol metabolism relative enzyme gene polymorphic test chip according to claim 1, it is characterized in that: said carrier is a slide.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410017946 CN1268769C (en) | 2004-04-21 | 2004-04-21 | Polymorphism detection chip for gene of enzyme relevant to ethanol metabolism, preparation method of usage |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410017946 CN1268769C (en) | 2004-04-21 | 2004-04-21 | Polymorphism detection chip for gene of enzyme relevant to ethanol metabolism, preparation method of usage |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1563421A CN1563421A (en) | 2005-01-12 |
| CN1268769C true CN1268769C (en) | 2006-08-09 |
Family
ID=34479252
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200410017946 Expired - Fee Related CN1268769C (en) | 2004-04-21 | 2004-04-21 | Polymorphism detection chip for gene of enzyme relevant to ethanol metabolism, preparation method of usage |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1268769C (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2324128A1 (en) * | 2005-09-29 | 2009-07-30 | Proyecto De Biomedicina Cima, S.L. | Molecular markers of hepatocellular carcinoma and their applications |
| JPWO2011043365A1 (en) * | 2009-10-07 | 2013-03-04 | 学校法人武庫川学院 | Genotyping method |
| CN102146438A (en) * | 2010-12-22 | 2011-08-10 | 协和干细胞基因工程有限公司 | Kit for detecting alcoholic liver disease susceptibility |
| CN103361432A (en) * | 2013-07-26 | 2013-10-23 | 天津市秀鹏生物技术开发有限公司 | Primer group and kit for detecting genetic typing of human aldehyde dehydrogenase 2 (ALDH2) |
| CN103540675A (en) * | 2013-10-31 | 2014-01-29 | 上海百傲科技股份有限公司 | Specific primer pair and probe for detecting ALDH2 (alcohol dehydrogenase 2) gene chip |
| 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 |
| CN104156632A (en) * | 2014-07-21 | 2014-11-19 | 金华市中心医院 | Method for intelligently designing drug metabolic enzyme detecting gene chip probes |
| CN113774115A (en) * | 2021-08-04 | 2021-12-10 | 上海百傲科技股份有限公司 | Method, kit, primer pair, probe, gene chip and application for detecting ALDH2 gene |
-
2004
- 2004-04-21 CN CN 200410017946 patent/CN1268769C/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN1563421A (en) | 2005-01-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1280422C (en) | Asymmetrical PCR amplification method, dedicated primer and use thereof | |
| EP0975807B1 (en) | Two-step hybridization and capture of a polynucleotide | |
| KR101072900B1 (en) | Method for selective labeling and detection of target nucleic acids using immobilized peptide nucleic acid probes | |
| JP2002538785A (en) | Customized oligonucleotide microchips that convert multigene information into simpler patterns are portable and reusable | |
| EP1612282B1 (en) | Probe set and substrate for detecting nucleic acid | |
| CN101034061A (en) | Method for detecting mononucleotide polymorphism with biochip | |
| CN109182501B (en) | Folate metabolism gene polymorphism detection primer and kit | |
| CN113278717B (en) | Primer pool, kit and method for detecting blood flow infection by targeted sequencing method | |
| CN1268769C (en) | Polymorphism detection chip for gene of enzyme relevant to ethanol metabolism, preparation method of usage | |
| CN1195070C (en) | Detection type gene chip for detecting various infectious desease and use thereof | |
| CN110938673A (en) | Method for generating single-stranded DNA (deoxyribonucleic acid) by using strand displacement primer mediated asymmetric PCR (polymerase chain reaction) | |
| CN101067149B (en) | CYP3A detecting chip and its application | |
| CN101168773B (en) | Nucleic acid sequencing method based on fluorescence quenching | |
| CN114427001A (en) | Kit for evaluating effectiveness of adalimumab in treating psoriasis based on 78 SNP loci | |
| CN110564861A (en) | Fluorescence labeling composite amplification kit for human Y chromosome STR locus and InDel locus and application thereof | |
| CN1814794A (en) | Leptin and leptin receptor gene polymorphism detecting chip, and its preparing method and use | |
| Bang‐Ce et al. | Rapid detection of common Chinese glucose‐6‐phosphate dehydrogenase (G6PD) mutations by microarray‐based assay | |
| KR100818276B1 (en) | Primer set probe set method and kit for predicting alcohol-degrading ability and hangover development | |
| CN111172273A (en) | Primer group, kit and detection method for SMN1 gene detection | |
| CN1865934A (en) | Method for constant temperature amplification and detection of nucleic acid signal by using molecular beacon | |
| US20060240430A1 (en) | Method for hybridisation of immobilized genomic dna | |
| CN112779322A (en) | Gene mutation detection kit based on non-fluorescence labeled probe and high-resolution melting curve, detection method and application thereof | |
| WO1997010366A2 (en) | High throughput screening method for sequences or genetic alterations in nucleic acids | |
| CN1289691C (en) | Quick detection method of pathogenic microbe diagnosis type gene chip | |
| CN110819708A (en) | Method for detecting ALDH2 gene polymorphism by fluorescent quantitative PCR |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20060809 |