CN101851672A - Method for simultaneously detecting 32 single nucleotide polymorphism (SNP) locus genotypes - Google Patents
Method for simultaneously detecting 32 single nucleotide polymorphism (SNP) locus genotypes Download PDFInfo
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Abstract
The invention relates to a method for simultaneously detecting 32 labeled single nucleotide polymorphism (SNP) loci. In the method, adducin-1, platelet endothelial cell adhesion molecule-1 (PECAM-1), C-reactive protein (CRP), endothelial constitutive nitric oxide synthase (ecNOS), plasma cell membrane glycoprotein-1 (PC-1), L-selectin, G-protein beta3 subunit gene, angiotensin I converting enzyme (ACE), angiotensin II type 1 receptor gene, proangiotensin, methylenetetrahydrofolate reductase (MTHFR) and hepatic lipase gene which are published on an international haplotype diagram engineering database are selected for detecting 32 labeled single nucleotide polymorphisms of the Han nationality in China; three primers are designed for each single nucleotide polymorphism (two PCR amplification primers and one extension primer); and the single nucleotide polymorphism loci are subjected to genotyping through polymerase chain reaction (PCR) amplification, extension and hybridization and by applying an SNP stream genotyping system. The detection method has the advantages of quickness, accuracy, high throughput, simple operation, microscale and the like.
Description
Technical field
The present invention relates to a kind of method that detects 32 mononucleotide polymorphism sites simultaneously.Belong to the Medical Molecular Biology field.
Background technology
In recent years, along with the raising of people's living standard and the change of mode of life, coronary heart disease has become the principal disease of current harm China people health and lives, and its morbidity is ascendant trend year by year, and mortality ratio surpasses tumour and leaps to the first.Its morbidity is the coefficient result of inherited genetic factors and environmental factors.The generation of coronary heart disease mainly causes owing to atherosclerotic plaque.Atherosclerotic formation is subjected to the influence of many-sided factor.A large amount of clinical experiments confirms that blood lipid level is playing vital role aspect atherosclerotic generation and the development.Blood fat disorder becomes coronary heart disease and atherosclerotic primary hazard factor.In addition, inflammatory reaction also runs through atherosclerotic whole process, thereby the activation of inflammatory reaction may be to cause the unstable principal element that causes acute coronary syndrome of patch.Think at present in the blood vessel that (endothelial function is impaired, oxidative stress etc.) and blood vessel outer (infection etc.) factor cause the generation of inflammatory reaction, inflammatory reaction causes production of cytokines again, further induces the expression of the effector molecule that phase reaction thing and some inflammatory responses are relevant when acute.
Along with science and technology development, clinically the coronary heart disease diagnosis means are gradually improved.Early diagnosis or eliminating coronary heart disease are of crucial importance to its prognosis.Electrocardiogram(ECG is the conventional means of cardiovascular disease diagnosis, treatment, it have easy, quick, inexpensive, do not have advantages such as wound and good reproducibility, but its susceptibility is poor, the false positive rate height, easily fails to pinpoint a disease in diagnosis, mistaken diagnosis.Two dimensional echocardiogram, radionuclide myocardial perfusion imaging, electron beam ct etc. have higher susceptibility and specificity to coronary heart disease diagnosis, but instrument costs an arm and a leg, though and the coronary angiography art is the gold standard of diagnosis of coronary heart disease, because of its technical sophistication, and have certain traumatic, thereby also difficult popularizing.
Along with the progress of molecular biology theory and Protocols in Molecular Biology, the gene mechanism of coronary heart disease and the applied research of gene test in diagnosis of coronary heart disease come into one's own day by day.Can be caused the polymorphism of dna sequence dna on the genomic level by the variation of single Nucleotide, this is the major cause that causes hereditary difference between individuality.The polymorphism of this mononucleotide has caused human difference at aspects such as disease susceptibilities.The frequency of variation in population as Nucleotide is called single nucleotide polymorphism greater than 1%.Known 1,500 ten thousand mononucleotide polymorphism sites, base mutation of promptly average per 300～600bp existence of in human genome, approximately existing.These mononucleotide polymorphism sites are the basic substance of species diversity (comprising population level and individual level) just, also is the basic substance of ill risk and medicine differential responses between polygene complex disease and individuality.Haplotype is to be positioned on the karyomit(e) or one group of mononucleotide polymorphism site that is associated in a certain zone.The genotype frequency of single nucleotide polymorphism, gene frequency and haplotype frequency all may with disease-related.Tagged single-nucleotide polymorphic is meant the mononucleotide polymorphism site that can represent other numerous site informations of chromosomal region.Therefore study tagged single-nucleotide polymorphic, most genetic polymorphism pattern in this zone can be provided, the validity of association analysis greatly is provided.Detect single nucleotide polymorphism and carry out the correlation analysis of disease, can single base level be brought up in the gene diagnosis of disease from the angle of heredity.Single nucleotide polymorphism also more and more receives publicity in the research of heredopathia research, population genetics, pharmacogenomics, medical jurisprudence, drug development and complex disease as the most general a kind of heritable variation type.
Along with going deep into of single nucleotide polymorphism research, Chinese scholars has been found a plurality of and the closely-related mononucleotide polymorphism site of coronary disease susceptibility.Adducin 1 is a kind of cytolemma skelemin, and there is the G614T point mutation in adducin 1 gene the 10th exon, and the glycine (Gly) of the 460th of aminoacid sequence is replaced by tryptophane (Trp).Discover not only closely related (the Alanna C.M of Gly460Trp polymorphism of adducin 1 gene with coronary heart disease, Molly S.B, Aaron R.F, et al.ADD1 460W allele associated with cardiovascular disease in hypertensive individuals.Hypertension, 2002,39 (6): 1053-1057), but also it is closely related with the risk factor hypertension and the Aorta elasticity of coronary heart disease, be the desirable early warning factor (the Yan L of Incidence of CHD and mortality ratio after the hypertension, Lutgarde T, Tatiana K, et al.Cardiovascular risk inrelation to a-Adducin Gly460Trp polymorphism and systolic pressure.Hypertension, 2005,46 (3): 527-532).
Thrombocyte endothelial cell adhesion molecule-the 1st, a kind of cell adhesion molecule, in inflammatory process, thrombocyte endothelial cell adhesion molecule-1 may be induced the initial phase of inflammation, and its expression can promote leukocytic migration.Thrombocyte endothelial cell adhesion molecule-1 gene has a plurality of single nucleotide polymorphism, wherein exon 2 transcription initiation site downstream the 373rd Nucleotide is replaced by guanine (G) by cytosine(Cyt) (C), causes coded product the 125th amino acids to be Xie Ansuan (Val) by leucine (Leu) variation.Domestic result of study shows the G373C polymorphism and the closely related (Chang Zhitang of coronary heart disease of thrombocyte endothelial cell adhesion molecule-1, Chen Jian, Cheng Longxian etc., the relation of thrombocyte endothelial cell adhesion molecule-1 gene Leu 125 Val polymorphisms and coronary heart disease and Hazard Factor, the old cardiovascular and cerebrovascular disease magazine of China, 2007,9 (10): 664-667).External result of study also points out the G373C polymorphism and the coronary heart disease of thrombocyte endothelial cell adhesion molecule-1 closely related, and the G1688A polymorphism of thrombocyte endothelial cell adhesion molecule-1 does not have obviously relevant (Lu Fang with coronary heart disease, Heming Wei, Sanual H.C, et al.Association of Leu125Val polymorphism of platelet endothelial celladhesion molecule-1 (PECAM-1) gene ﹠amp; Soluble level of PECAM-1with coronary artery disease in Asian Indians.Indian J Med Res, 2005,121 (2): 92-99; Wei H, Fang L, Chowdhury SH, et al.Platelet-endothelial cell adhesionmolecule-1gene polymorphism and its soluble level are associated with severe coronary artery steecNOSisin Chinese Singaporean.Clin Biochem, 2004,37 (12): 1091-1097).Patients with coronary heart disease serum thrombocyte endothelial cell adhesion molecule-1 level obviously raises, and relevant with the genotype of G373C polymorphism; Serum thrombocyte endothelial cell adhesion molecule-1 level also with serum in palatelet-selectin concentration, the peripheral blood platelet count is closely related with leukocyte count, prompting patients with coronary heart disease inflammation is the important factor that thrombocyte endothelial cell adhesion molecule-1 level raises.
Change of serum C-reactive protein is the mark of inflammatory reaction.The C-reactive protein not only can be predicted the risk of incidence of coronary heart disease and generation, also can assess (Roy D to the prognosis of coronary heart disease, Quiles J, Avanzas P, et al.A comparative study of markersof inflammation for the assessment of cardiovascular risk in patients presenting to the emergency departmentwith acute chest pain suggestive of acute coronary syndrome.Int J Cardiol, 2006,109 (3): 317-321).Ding Yanping etc. study the 1059G/C gene pleiomorphism of C-reactive protein, find that GG genotype C-reactive protein level is significantly higher than the GC+CC genotype, do not find that 1059G/C gene pleiomorphism and coronary heart disease take place and the dependency (Ding Yanping of severity degree of coronary, Ma Zhoujian, the correlation research of c reactive protein and gene pleiomorphism thereof and incidence of coronary heart disease, Shandong medicine, 2006,46 (19): 36-37).
Nitrogen protoxide is a kind of important biological molecule; participate in a series of physiological activities such as vasodilation in the control agent, vascular permeability, platelet adhesion reaction and gathering, nerve signal transmission, host defense; be anti-coronary atherosclerosis and thrombosis in the cardiovascular systems, keep the easypro requisite protection factor of reaction that contracts of normal blood vessels.Endothelium own type nitricoxide synthase has important regulation as synthetic nitric oxide production key enzyme to nitrogen protoxide, and its gene morphs, and can influence proteic function of endothelium own type nitricoxide synthase and activity.Relevant (the Tangurek B of a plurality of single nucleotide polymorphism that bibliographical information endothelium own type nitricoxide synthase is arranged with cause of coronary heart disease, Ozer N, Sayar N, et al.The relationshipbetween endothelial nitric oxide synthase gene polymorphism (T-786C) and coronary artery disease in theTurkish population.Heart Vessels, 2006,21 (5): 285-290; Lin NT, Lee MJ, Lee RP, et al.Analysis of endothelialnitric oxide synthase gene polymorphisms with cardiovascular diseases in eastern Taiwan.Chin J Physiol, 2008,51 (1): 42-47).
Plasmocyte membrane glycoprotein 1 gene is the active inhibition of insulin receptor tyrosine kinase, can suppress the insulin receptor tyrosine kinase activity, weakens the biological action of Regular Insulin, thereby is one of candidate gene of diabetes B.Diabetes and insulin resistant are the independent hazard factors of coronary heart disease.Discover, the K121Q polymorphism of plasmocyte membrane glycoprotein 1 gene and closely related (the Simonetta B of the generation of diabetes and coronary heart disease, Ornella L, Sabrina P, et al.The K121Q polymorphism ofthe ENPP1/PC-1 gene is associated with insulin resistance/atherogenic phenotypes, including earlier onsetof type 2diabetes and myocardial infarction.Diabetes, 2005,54 (10): 3021-3025).
G albumen claims G-protein again, is the signal transmission factor between cell-membrane receptor and the cell internal effect device, signal conduction in the cell after the excitement of mediation g protein coupled receptor, the propagation and the atomization of participation cell.G albumen plays a part very important to integrity and the adjusting antiotasis of keeping vessel wall.But the propagation of the activation stimulated vascular smooth muscle cell of G albumen approach signal conduction.Result of study shows that the G protein ' beta ' 3 C825T of subunit polymorphic allele is not only relevant with essential hypertension, but also with relevant (the Von B of lesion degree of coronary heart disease and coronary artery, Schusterschitz Y, Koch W, et al.G proteinbeta 3subunit 825T allele carriage and risk of coronary artery disease.Atherosclerosis, 2003,167 (1): 135-139).
L-selects plain membranin as a kind of high glycosylation, mainly be expressed in neutrophil leucocyte, monocyte and some lymphocyte subgroup surface, select element and the palatelet-selectin that is expressed in activated blood platelet, endothelial cell surface to constitute jointly with the E-that is expressed in the activation endothelial cell surface and select plain family.L-selects element to participate in processes such as leukocytic activation, inflammation and wound, make white corpuscle roll, move and stick along endothelial cell surface, and by integrating the plain booster action that sticks with β 2, make leukocyte activation, be out of shape, pass through vascular endothelial cell and advance under people's endocardium, discharge the inflammatory mediator inducing inflammatory reaction.L-selects element and palatelet-selectin to discern mutually simultaneously, can trigger white corpuscle and combine with thrombocyte, and haemodynamics changes, and the blood flow stasis of blood stagnates, and forms thrombus easily, increases the weight of myocardial ischemia.Result of study shows that Chinese han population L-selects plain gene P213S polymorphism relevant with coronary heart disease.CHD group serum L-selects plain level obviously to raise.Prompting L-selects element to participate in coronary heart disease generation, evolution (Xia Zunen, Li Yan, bear Junichiro Koizumi etc.Aged coronary heart disease patient L-selects plain P213S Study on gene polymorphism.China's gerontology magazine, 2006,26 (5): 594-596).
Renin-angiotensin system plays crucial effect in keeping body water salt balance and adjusting cardiovascular activity.Angiotensin i-converting enzyme is the key enzyme of renin-angiotensin system, can change nonactive type Angiotensin into active type and deactivation kassinin kinin, play an important role aspect body water-electrolyte metabolism, homeostasis and the adjusting cardiovascular function keeping, its effect in hypertension incidence has obtained certainly.The angiotensin i-converting enzyme gene is positioned at long-armed 2 districts of karyomit(e) 3 bands No. 17, is made up of 26 exons and 25 introns.Discover, being present in size of angiotensin i-converting enzyme gene 16 introns is insertion/disappearance (I/D) polymorphism and plasma angiotensinogen I conversion enzyme level and relevant (the Acarturk E of cause of coronary heart disease of 287bp, Attila G, Bozkurt A, et al.Insertion/deletion polymorphismof the angiotensin converting enzyme gene in coronary artery disease in southern Turkey.J Biochem MolBiol, 2005,38 (4): 486-490).In renin-angiotensin system, the effect of Angiotensin II is the strongest.Angiotensin II has the vasoconstriction effect and is responsible for regulating water-electrolyte metabolism, aldosterone biosynthesizing.But also stimulated vascular smooth muscle cell growth of Angiotensin II, myocardial cell and intimal hyperplasia improve sympathetic activity, increase coronary resistance, even may bring out irregular pulse.Angiotensin II mainly plays a role by combining with Angiotensin II 1 receptor.Angiotensin II 1 receptor causes water-sodium retention and elevation of blood pressure after activating, and is the key link that renin-angiotensin system acts on effector.Domestic scholars is analyzed a plurality of single nucleotide polymorphism of Angiotensin II 1 receptor and the dependency of coronary heart disease, but the result is not quite similar.Proangiotensin is the substrate of feritin, forms Angiotensin II under the effect of feritin and angiotensin-converting enzyme.Result of study shows the G-6A of angiotensinogen gene and the closely related (Yang Fan of coronary disease susceptibility of M235T polymorphism and Chinese han population, Zhao Luosha, Zheng Hong etc., the correlation research of angiotensinogen gene G-6A polymorphism and coronary heart disease, Shandong medicine, 2008,48 (1): 19-21; Appoint clean, Jia Yongping, Lv Jiyuan etc., the correlation research of angiotensinogen gene M235T polymorphism and coronary heart disease, Mountain Western Medicine S University's journal, 2005,36 (4): 439-442).
The homocysteine mass formed by blood stasis may cause atherosclerosis by number of mechanisms, comprise and cause endothelial injury and dysfunction, stimulated vascular smooth muscle cell hyperplasia, destroy body blood coagulation and fibrinolytic system, influence lipid metabolism etc., make body be in the preceding state of thrombus, thereby increased the danger of cardiovascular and cerebrovascular diseases morbidity.Hyperhomocysteinemiainjury is cardiovascular and cerebrovascular diseases independent risk factor such as atherosclerosis and thrombosis.In vivo, methionine(Met) generates homocysteine through series reaction such as demethylations, the homocysteine that generates has 50% approximately under the effect of methionine synthase, with the vitamin B12 is cofactor, with the N5-methyl tetrahydrofolate is methyl donor, takes place to methylate, again synthetic methionine again, methyl donor in this reaction is by Methylene tetrahydrofolate reductase catalyzing N 5, N10-methylene tetrahydrofolate and producing.So Methylene tetrahydrofolate reductase is a key enzyme in the methionine(Met) metabolism.The transgenation of Methylene tetrahydrofolate reductase can cause enzymic activity to reduce, and causes folic acid and vitamin B12 metabolic disturbance, and then influence homocysteine and be converted into methionine(Met), and proper interior homocysteine level is increased.The C677T polymorphism of result of study demonstration Methylene tetrahydrofolate reductase gene is distributed between CHD group and the control group and has significant difference, and with closely related (Mager A of cause of coronary heart disease age, Koren-Morag N, Shohat M, et al.Impact of ethnicity and MTHFR genotype on age at ohset of coronaryartery di sease in women in Israel.Isr Med Assoc J, 2008,10 (7): 516-519), but with irrelevant (the Jos é M.G P of the prognosis of coronary heart disease, Salvador E C, Manuel J N, et al.Influence of 677 C → T polymorphism of methylenetetrahydrofolatereductase on medium-term progecNOSis after acute coronary syndromes.Tex Heart Inst J, 2007,34 (2): 142-147).By the individual Plasma Homocysteine of its 3 kinds of different genotype is detected, the result shows that the Plasma Homocysteine of CHD group is apparently higher than control group, no matter be in CHD group or in control group, the homocysteine level of TT genotype individuality all is higher than TC genotype and the genotypic individuality of CC, and the difference between the homocysteine level of TC genotype and CC genotype individuality does not have significance in these two groups.In the correlation research to Methylene tetrahydrofolate reductase gene another polymorphism A1298C and coronary heart disease, the result who obtains at different national populations but is not quite similar.
Hepatic lipase is synthetic by hepatic parenchymal cells, is one of key enzyme important in the lipoprotein metabolism, can interior triglyceride level and the phosphatide of the various lipoprotein of hydrolysis.The hepatic lipase vigor can cause the plasma lipoprotein metabolism disorder unusually, with coronary heart disease certain dependency is arranged.By to hepatic lipase gene promoter-250G/A ,-514C/T ,-710T/C and-detection of 4 site single nucleotide polymorphism of 763A/G, the polymorphism of finding the hepatic lipase gene is relevant with coronary heart disease, and (Shi Jin is pretty, Ni Peihua, Zheng Shougui etc., hepatic lipase gene promoter 250G/A polymorphism and Coronary Heart Disease, China's laboratory medicine magazine, 2006,29 (6): 515-517; Hu Min, Shao Jianguo, Zhu Yi etc., hepatic lipase gene-514C/T polymorphism and coronary heart disease correlation research, Chinese Pharmacological circular, 2006,22 (9): 1118-1121.).
The detection method of single nucleotide polymorphism mainly contains order-checking, high performance liquid chromatography, restriction enzyme digestion, gene chip and real-time fluorescence quantitative polymerase chain reaction (PCR) etc. at present.These methods can only detect one or a few single nucleotide polymorphism, are not suitable for a plurality of single nucleotide polymorphism, the detection of broad variety single nucleotide polymorphism and sample in enormous quantities.Therefore select the adducin 1 announced on the international haplotype figure engineering data base, thrombocyte endothelial cell adhesion molecule-1, the C-reactive protein, endothelium own type nitricoxide synthase, plasmocyte membrane glycoprotein 1, L-selects plain, G protein ' beta ' 3 subunit, angiotensin i-converting enzyme, Angiotensin II 1 receptor, proangiotensin, the tagged single-nucleotide polymorphic of Methylene tetrahydrofolate reductase and hepatic lipase gene Chinese han population, set up the detection method of a plurality of single nucleotide polymorphism,, screen the single nucleotide polymorphism relevant with coronary heart disease by single nucleotide polymorphism is carried out gene type, a kind of new approaches are provided from the genetics level for the prediction and the control of cardiovascular risk factors, make people in time to predict and to estimate risk of cardiovascular diseases, for cardiovascular disorder early prevention and treatment in time provide guide of theory by to healthy population or occur the typing gene polymorphisms of cardiovascular risk factors colony in early days.
Summary of the invention
(1) technical problem that will solve
The purpose of this invention is to provide a kind of 12 methods with 32 Chinese han population tagged single-nucleotide polymorphic locis of coronary heart disease dependent genes that detect simultaneously.These genes and 32 tagged single-nucleotide polymorphic locis are adducin 1 gene: rs3775067, rs1263359, thrombocyte endothelial cell adhesion molecule-1 gene: rs503550, C-reactive protein gene: rs1205, endothelium own type nitric oxide synthase gene: rs7830, rs3918188, plasmocyte membrane glycoprotein 1 gene: rs12528076, rs858341, rs021966, rs858345, rs9308995, L-selects plain gene: rs964555, rs2205848, rs4987318, rs2298900, G protein ' beta ' 3 subunit gene: rs5445, angiotensin i-converting enzyme gene: rs4333, rs4305, rs4353, Angiotensin II 1 receptor gene: rs6801836, rs2675511, rs5182, angiotensinogen gene: rs1926722, rs7539020, rs3889728, rs493132, rs478523, Methylene tetrahydrofolate reductase gene: rs1801133, rs9651118, rs6541003 and hepatic lipase gene: rs12462668, rs10426971.
(2) technical scheme
The ultimate principle of the method for the invention is single-basic extension and label microarray.The single-basic extension method claims little sequencing again, is the method for detecting single nucleotide polymorphism that development is come out on the basis of dideoxy sequencing method.At first the method with PCR amplifies the fragment that contains the purpose mononucleotide polymorphism site from genomic dna, carry out " sequencing reaction " with a primer that is complementary with the mononucleotide polymorphism site upstream sequence, and in reaction, add fluorescently-labeled dideoxy nucleotide, making this " sequencing reaction " measure mononucleotide polymorphism site is just to have stopped after which base.SNP Stream gene type system is a cover high automation, high-throughout single nucleotide polymorphism classification system, it combines multiple PCR technique and fluorescent mark single-basic extension typing method, simultaneously oligonucleotide microarray technique is applied in the 384 conventional orifice plates, and introducing microarray hybridization technology, the result can be read by the Two Colour Fluorescence on the imager, this system can carry out sample rapid detection (Wang IJ in enormous quantities to 48 mononucleotide polymorphism sites of 4 types of as many as simultaneously, Chiang TH, Shih YF, etal.The association of single nucleotide polymorphisms in the MMP-9genes with susceptibi lity to acute primaryangle closure glaucoma in Taiwanese patients.Molecular Vision, 2006,12:1223-1232; Pollin TI, Tanner K, Connell JR, et al.Linkage of plasma adiponectin levels to 3q27explained by association with variation inthe APM1gene.Diabetes, 2005,54:268-274; Damcott CM, Ott SH, Pollin TI, et al.Genetic variation inadiponectin receptor 1and adiponectin receptor 2is associated with type 2diabetes in the old order amish.Diabetes, 2005,54:2245-2250).
Method of the present invention, it comprises the steps:
(1) is designed for 32 the extension primers that contain the segmental 32 pairs of primers of purpose mononucleotide polymorphism site and order-checking and hybridization that increase and sees Table 1.
32 of the 32 pairs of primers that table 1 the present invention is designed and order-checking and hybridization are extended primers
(2) dilution and mix the PCR primer to make its final concentration be 2.5 μ M makes up PCR primer pond and the purpose fragment is increased.Per 96 person-portion PCR reaction systems are:
Reagent becomes partial volume (μ L)
Primer pond (2.5 μ M) 11.2
Deoxynucleotide (2.5mM) 20
10 times of PCR damping fluid II 56.2
Magnesium chloride (25mM) 112.6
Gold medal amplification enzyme (5U/ μ L) 11.2
Distilled water 126
The PCR cycling condition is:
(3) amplified production is carried out purifying.The preparation of refined solution:
Reagent becomes partial volume (μ L)
Excision enzyme I (20U/ μ L) 11.2
Shrimp alkaline phosphotase (1U/ μ L) 112
10 times of shrimp alkaline phosphotase damping fluids 34
Distilled water 180
The condition of purification reaction is:
(4) dilution and mix to extend primer to make its final concentration be 5 μ M makes up and extends the primer pond, carries out extension.Per 96 person-portion PCR reaction systems are:
Reagent becomes partial volume (μ L)
Extend dilution buffer liquid 423
Extend primer pond 3.4
20 times extend dideoxy nucleotides (T/C and A/G) each 11.3
Distilled water 334
Archaeal dna polymerase 2.36
The condition of extension is:
(5) hybridization.In extension products, add 8 μ L hybridization solutions (50 μ L hybridizing reagents are added in the 850 μ L hybridization buffers), get 15 μ L behind the mixing and add in the hybridization plate reacting hole 42 ℃ of hybridization 2h.
(6) the hybridization plate is carried out fluorescent scanning, judge the genotype of mononucleotide polymorphism site according to fluorescent signal.
Description of drawings
The single nucleotide polymorphism scanning result of Fig. 1 sample is through SNP Stream gene type system scan, can obtain the scanning result of three kinds of colors, be homozygous blueness (XXL=0.95, XXU=1.0) or green (YYL=0.02, YYU=0.06), heterozygous orange (XYL=0.38, XYU=0.7), reference line (baseline=2.22).
Beneficial effect of the present invention
Method of the present invention is used Single base extension and label microarray principle, can detect simultaneously the genotype of 32 mononucleotide polymorphism sites in same system, have fast, accurately, the advantages such as high flux, simple to operate, traceization.
The used deoxynucleotide of experiment is available from Japanese TaKaRa company among the present invention, PCR buffer solution II, magnesium chloride, gold medal amplification enzyme are available from Switzerland Roche company, excision enzyme I is available from New England BioLabs company, shrimp alkaline phosphatase and buffer solution thereof, sterilization distilled water be available from U.S. Promega company, other reagent, solution, hybridization plate etc. except indicating the source all available from U.S. Beckman Coulter company.
Embodiment
Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.
Embodiment 1 usefulness method of the present invention detects the genotype of 32 mononucleotide polymorphism sites of Chinese han population simultaneously.
(1) is designed for 32 extension primers that amplification contains the pulsating 32 pairs of primers of purpose mononucleotide polymorphism site and order-checking and hybridization, sees sequence 1 for details to sequence 96.。
(2) dilution and mix the PCR primer to make its final concentration be 2.5 μ M makes up PCR primer pond and the purpose fragment is increased.Per 96 person-portion PCR reaction systems are:
Reagent becomes partial volume (μ L)
Primer pond (2.5 μ M) 11.2
Deoxynucleotide (2.5mM) 20
10 times of PCR damping fluid II 56.2
Magnesium chloride (25mM) 112.6
Gold medal amplification enzyme (5U/ μ L) 11.2
Distilled water 126
The PCR cycling condition is:
(3) amplified production is carried out purifying.The preparation of refined solution:
Reagent becomes partial volume (μ L)
Excision enzyme I (20U/ μ L) 11.2
Shrimp alkaline phosphotase (1U/ μ L) 112
10 times of shrimp alkaline phosphotase damping fluids 34
Distilled water 180
The condition of purification reaction is:
(4) dilution and mix to extend primer to make its final concentration be 5 μ M makes up and extends the primer pond, carries out extension.Per 96 person-portion PCR reaction systems are:
Reagent becomes partial volume (μ L)
Extend dilution buffer liquid 423
Extend primer pond 3.4
20 times extend dideoxy nucleotides (T/C and A/G) each 11.3
Distilled water 334
Archaeal dna polymerase 2.36
The condition of extension is:
(5) hybridization.In extension products, add 8 μ L hybridization solutions (50 μ L hybridizing reagents are added in the 850 μ L hybridization buffers), get 15 μ L behind the mixing and add in the hybridization plate reacting hole 42 ℃ of hybridization 2h.1000 leave the heart dries.The dcq buffer liquid of drawing after 20 μ L dilute 2 (60 times of dcq buffer liquid 2) joins in each hole of hybridizing on the plate, and centrifuge dripping cleans altogether 3 times.
(6) scanning: input and hybridization each reacting hole of plate corresponding sample message in SNPstream V2.2 software (Beckman Coulter company), import the primer sites file, scan and hybridize behind the plate by this software analysis sweep signal and derive the result.
Sequence table
＜110〉Chinese People's Liberation Army General Hospital
＜120〉a kind ofly detect 32 genotypic methods of mononucleotide polymorphism site simultaneously
<160>96
<210>1
<211>20
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs6541003 SNP site:
<400>1
CACTGATCAT?CCGAATCACC 20
<210>2
<211>22
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs6541003 SNP site:
<400>2
TTTTTCAAAA?AGTGGATCTC?CA 22
<210>3
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs6541003 SNP site:
<400>3
CTCACTATCT?GACAAGCCAC?GCTCAAGAAG?TAAAACACAC?ATTCC 45
<210>4
<211>18
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs7503550 SNP site:
<400>4
AAGGCTGAGG?ACCTGGCC 18
<210>5
<211>25<212>DNA
＜213〉artificial sequence
<220>
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs7503550 SNP site:
<400>5
ATTATGTGAT?CTGTAGTTTG?GAAGC 25
<210>6
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs7503550 SNP site:
<400>6
CTAACTAAGC?TACGCCGACA?TTCCTCCCGA?TCCCCTCCAA?ACCCA
<210>7
<211>27
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs3775067 SNP site:
<400>7
TAACCAATTC?AAACTTATTT?AATCAGC 27
<210>8
<211>23
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs3775067 SNP site:
<400>8
TGTGGTGGAG?ATTAGTGCTA?AGT 23
<210>9
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs3775067 SNP site:
<400>9
CACCGCTATC?AACAGACTTG?CCAATCACAG?CTGTAACATT?TTCAC 45
<210>10
<211>23
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs1926722 SNP site:
<400>10
TGTAGGTTTT?GCTGAAATTT?TCC
<210>11
<211>18
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs1926722 SNP site:
<400>11
CCAGGCAGGC?TTATGCTC 18
<210>12
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs1926722 SNP site:
<400>12
CACGACAAGA?CAACAGATAC?GGATGACTTA?GTTGGGTGAT?GGGGG 45
<210>13
<211>21
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs1205 SNP site:
<400>13
TCTGTTGTTT?GTCAATCCCT?T 21
<210>14
<211>21
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs1205 SNP site:
<400>14
ATCTTCTTGC?TGCTGGATTT?C 21
<210>15
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs1205 SNP site:
<400>15
ACGTAAGACC?ACTCAAGACC?ACTTCCAGTT?TGGCTTCTGT?CCTCA 45
<210>16
<211>18
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs7539020 SNP site:
<400>16
ACCAGGCCCC?CTGACTCT 18
<210>17
<211>22
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs7539020 SNP site:
<400>17
GGGATAAGCT?AAAAGGCAGA?TT 22
<210>18
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs7539020 SNP site:
<400>18
ACCGCACTAA?GCAATGTATC?ATCCACGAGC?TGAAGCACCA?ATCTA 45
<210>19
<211>28
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs5445 SNP site:
<400>19
ATCTCATTCA?GGTGTTCTCT?TCTATATT 28
<210>20
<211>18
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs5445 SNP site:
<400>20
TGTCCCTGAT?GCTGCCTC 18
<210>21
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs5445 SNP site:
<400>21
CACTAGTCAT?AACGCAGCCT?GGGTGCCATT?CCCACTAAGC?TTTCT 45
<210>22
<211>18
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs3889728 SNP site:
<400>22
GAGGGAAAAG?GGGAGCTG 18
<210>23
<211>18
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs3889728 SNP site:
<400>23
TGAGGTTTCC?CTGATGCA 18
<210>24
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs3889728 SNP site:
<400>24
CAGTCAACAA?TCCAGATCAA?GAGAGAGTTG?AGAGTGCCCG?GTGTG 45
<210>25
<211>22
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs2493132 SNP site:
<400>25
TAAACTAAAA?AAGGGAAGCT?GC 22
<210>26
<211>18
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs2493132 SNP site:
<400>26
TCGACAGATT?TACCTAGCCT?CC 22
<210>27
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs2493132 SNP site:
<400>27
CAGAACATCC?TCAGAAGCAA?CCGTCTTCCC?TCTGCTCGTC?TGACA 45
<210>28
<211>20
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs7830 SNP site:
<400>28
CCTCTGTCCC?TAGATTGTGT 20
<210>29
<211>18
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs7830 SNP site:
<400>29
ATTCTGGCAG?GAGCGGCT 18
<210>30
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs7830 SNP site:
<400>30
GCAAGCCATC?AGCTAATACA?ACTCCCTTCA?GGCAGTCCTT?TAGTC 45
<210>31
<211>20
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs3918188 SNP site:
<400>31
CCCAGCTGAA?GCATTTAAAA 20
<210>32
<211>18
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs3918188 SNP site:
<400>32
TGAAGGGCAG?CTCGGTGG 18
<210>33
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs3918188 SNP site:
<400>33
CGCAGAAGCA?ACTCACTTCT?TGGGAGCAAG?GCACACGTAC?AAGGG 45
<210>34
<211>18
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs4353 SNP site:
<400>34
ATGAGATGCA?GAATCGCC 18
<210>35
<211>18
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs4353 SNP site:
<400>35
ATTGTGTACT?TTCTAGAGAA?TGTGTGG 27
<210>36
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs4353 SNP site:
<400>36
AGACCGACAA?GCAATCTACA?TGTACACATG?CTTTATCTCC?CAAGA 45
<210>37
<211>19
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs1801133 SNP site:
<400>37
AGCTTTGAGG?CTGACCTGA 19
<210>38
<211>18
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs1801133 SNP site:
<400>38
ATGTCGGTGC?ATGCCTTC 18
<210>39
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs1801133 SNP site:
<400>39
CCATAACAAC?TTACCAGCCA?TTGAAGGAGA?AGGTGTCTGC?GGGAG 45
<210>40
<211>19
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs12528076 SNP site:
<400>40
AACGCAAGAA?AGTAGGGCT 19
<210>41
<211>24
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs12528076 SNP site:
<400>41
TCTAGAGGGT?CTCCCACTAG?TTTA 24
<210>42
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs12528076 SNP site:
<400>42
AGTAGCCTAA?CAGCACTCGA?GCTGCAATGC?TCAGTGGATG?TGATG 45
<210>43
<211>18
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs4305 SNP site:
<400>43
AAACAAGCAC?TGTGGCCC 18
<210>44
<211>18
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs4305 SNP site:
<400>44
ATGTCCTGGG?GCTGCAAG 18
<210>45
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs4305 SNP site:
<400>45
GCAACATAAG?ACCGCTCAAC?ATGCTGCCAC?TGTCATTTCT?GGCTG 45
<210>46
<211>20
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs10426971 SNP site:
<400>46
TGCTGAGACC?AAAGGTAAAT 20
<210>47
<211>26
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs10426971 SNP site:
<400>47
TTCTTCTGAA?AAGTAATCAC?TTGGTA 26
<210>48
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs10426971 SNP site:
<400>48
GATCCATCAA?CAGACATCAC?CACTGCAGCA?GAGCACTGCA?GTCCT 45
<210>49
<211>20
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs6801836 SNP site:
<400>46
AAATTCTTCT?CCCTTTTTCC?TC 22
<210>50
<211>26
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs6801836 SNP site:
<400>50
CCAATTAGAA?GAACATTTTA?CATAAATG 28
<210>51
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs6801836 SNP site:
<400>51
CCACTCAACT?CCACGAATAC?ATGACCAGAA?CTCCCTGCCC?AATAA 45
<210>52
<211>20
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs858341 SNP site:
<400>52
ATCCCATTGA?ATATACTTAA?TAAGATGTC 29
<210>53
<211>26
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs858341 SNP site:
<400>53
ATTGGAGGTA?AGCCTAAGAC?AAA 23
<210>54
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs858341 SNP site:
<400>54
AACATCCACG?CAACTCATAC?TTTCTATTCC?CCTCTCCCCT?TTACC 45
<210>55
<211>20
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs2021966 SNP site:
<400>55
AATCCTGAAA?ATAGTTGGTC?TCTT 24
<210>56
<211>26
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs2021966 SNP site:
<400>56
AGCTGAACTA?AACTTTACAA?TTCTTTTC 28
<210>57
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs2021966 SNP site:
<400>57
GCAGACAACG?AACAACTACC?TGCAGCAACT?ATCAAGAAAG?ATATG 45
<210>58
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs1263359 SNP site:
<400>58
AAATTCCAGA?CTGCAAATGG 20
<210>59
<211>26
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs1263359 SNP site:
<400>59
TCATCTAGAG?GCAGACATAC?TAGCT 25
<210>60
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs1263359 SNP site:
<400>60
CAACAATACG?AGCCAGCAAG?TAAGAAGGTC?TTATGTTTCC?AAAAT?45
<210>61
<211>26
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs2675511 SNP site:
<400>61
AAGATATAAT?GCATGAAATA?AAAGCC 26
<210>62
<211>25
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs2675511 SNP site:
<400>62
AATAGAGTTG?CTACTGAAAA?ACCCT 25
<210>63
<211>45
<212>DNA
＜213〉artificial sequence
<220>
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs2675511 SNP site:
<400>63
AGCAAGACCA?CCTAGACCAG?ACCTTTGATA?ATTTATATCT?CTCAC 45
<210>64
<211>27
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs5182 SNP site:
<400>64
ATTTTTCATT?GAGAACACCA?ATATTAC 27
<210>65
<211>25
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs5182 SNP site:
<400>65
ACAGGAAACC?CAGTATATTT?TTGG 24
<210>66
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs5182 SNP site:
<400>66
ACAACTCACG?CAAGTACCAT?ATTATGAGTC?CCAAAATTCA?ACCCT 45
<210>67
<211>23
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs9398995 SNP site:
<400>67
AGCTTCCAAG?CAAAAATTAG?AAT 23
<210>68
<211>24
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs9398995 SNP site:
<400>68
TTAAACAACA?TGAAAAAAAA?CCAC 24
<210>69
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs9398995 SNP site:
<400>69
CAACAAGTAA?TCCGCAGACT?ACAGCTTCCT?GATCCTTACA?CAAAA 45
<210>70
<211>23
<212>DNA
＜213〉artificial sequence
<220>
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs964555 SNP site:
<400>70
TAATGCCTAC?AGAATCTCAA?TGC 23
<210>71
<211>23
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs964555 SNP site:
<400>71
TTTCAGAGTT?CCAGTCATTG?AAC 23
<210>72
<211>45
<212>DNA
＜213〉artificial sequence
<220>
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs964555 SNP site:
<400>72
ACAATCAACA?TACGAACAGC?TGGTTCATAG?AGTTCCTGAA?AATGC 45
<210>73
<211>20
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs9651118 SNP site:
<400>73
TGTGTGAGAG?CAGCACTTCA 20
<210>74
<211>22
<212>DNA
＜213〉artificial sequence
<220>
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs9651118 SNP site:
<400>74
TTCCCCTAAC?TCTCAATCAT?GA 22
<210>75
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs9651118 SNP site:
<400>75
ATACCTACCA?CGCTACAGCC?ACTTTTCACA?GCGCTTGCCT?GTTTA 45
<210>76
<211>19
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs12462668 SNP site:
<400>76
TAGGGCTTCT?CTCCAGCAT 19
<210>77
<211>22
<212>DNA
＜213〉artificial sequence
<220>
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs12462668 SNP site:
<400>77
ACCCAACAAA?TGTGAAGAAT?GT 22
<210>78
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs12462668 SNP site:
<400>78
CCAGATCCTC?ACCATGTAAG?AATTGCCTTA?TGTGTAGTAA?GAGTC 45
<210>79
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs2205848 SNP site:
<400>79
TTTTATCCCA?ACACTGTGCT?T 21
<210>80
<211>22
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs2205848 SNP site:
<400>80
AGAAATGGGA?AGTTTTGATC?AG 22
<210>81
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs2205848 SNP site:
<400>81
ATCTAACGCA?CCTACGACCT?CTCAAAAAGA?CTTGTTGAGA?AGTGA 45
<210>82
<211>21
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs4987318 SNP site:
<400>82
AATCAGACCC?TCTGCCAATA?T 21
<210>83
<211>28
<212>DNA
＜213〉artificial sequence
<220>
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs4987318 SNP site:
<400>83
AGCCTCTATT?TGAAAATACT?GGTATAAA 28
<210>84
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs4987318 SNP site:
<400>84
CCGCCAGTAA?GACCTAGACG?AAAAAAGTAG?ATTCCTTCAA?ATGTT 45
<210>85
<211>21
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs2298900 SNP site:
<400>85
TGTCCATTTA?CACTGGCAAT?T 21
<210>86
<211>22
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs2298900 SNP site:
<400>86
TCACACACTT?TGTCTTTCTG?GA 22
<210>87
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs2298900 SNP site:
<400>87
AGACTTCTAC?GCAAGCACTG?TTCATTTAAA?TTTGAGAATA?GTGGG 45
<210>88
<211>24
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs858345 SNP site:
<400>88
TAAACAAAGG?TTTGAGTATC?TGCA 24
<210>89
<211>26
<212>DNA
＜213〉artificial sequence
<220>
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs858345 SNP site:
<400>89
AGAGTTGTTG?TTGCCTTTAT?TAGATT 26
<210>90
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs858345 SNP site:
<400>90
CAGCCATCCA?TTCACTATCT?AATACAAAAA?AAAAAAATAA?TTTGC 45
<210>91
<211>18
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs2478523 SNP site:
<400>91
TTGGGTGCAG?AGCAGGGC 18
<210>92
<211>24
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs2478523 SNP site:
<400>92
TTGAAAGACA?GACACTAACT?GGAG 24
<210>93
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs2478523 SNP site:
<400>93
CTCAGACTAC?GAATCCACGT?CCTGTGTGTC?TGTCTACCAG?TCCTC?45
<210>94
<211>18
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs4333 SNP site:
<400>94
TGAGAGCTCA?TGTGCAGG 18
<210>95
<211>21
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs4333 SNP site:
<400>95
AAAACAAGAA?AGGGCATTTC?C 21
<210>96
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs4333 SNP site:
<400>96
TACAAGCACG?CACTAGACAT?TGCTGGGTGA?GAGCACAGAG?TTGGG 45
Claims (4)
1. method that detects 32 mononucleotide polymorphism sites simultaneously, these genes and 32 tagged single-nucleotide polymorphic locis are adducin 1 gene: rs3775067, rs1263359, thrombocyte endothelial cell adhesion molecule-1 gene: rs503550, C-reactive protein gene: rs1205, endothelium own type nitric oxide synthase gene: rs7830, rs3918188, plasmocyte membrane glycoprotein 1 gene: rs12528076, rs858341, rs021966, rs858345, rs9308995, L-selects plain gene: rs964555, rs2205848, rs4987318, rs2298900, G protein ' beta ' 3 subunit gene: rs5445, angiotensin i-converting enzyme gene: rs4333, rs4305, rs4353, Angiotensin II 1 receptor gene: rs6801836, rs2675511, rs5182, angiotensinogen gene: rs1926722, rs7539020, rs3889728, rs493132, rs478523, Methylene tetrahydrofolate reductase gene: rs1801133, rs9651118, rs6541003 and hepatic lipase gene: rs12462668, rs10426971 is characterized in that detection method comprises the steps:
(1) sequence 1 that designs and synthesizes 32 mononucleotide polymorphism sites that are used to increase is to sequence 96 primers, 3 primers of each single nucleotide polymorphism design, and wherein 2 is the pcr amplification primer, is used to amplify the segment that contains the purpose mononucleotide polymorphism site; 1 for extending primer, be used for mononucleotide polymorphism site order-checking and with glass-chip on oligonucleotide chain hybridization;
(2) dilution and mixing PCR primer carry out pcr amplification to target dna;
(3) amplified production is carried out purifying;
(4) primer is extended in dilution and mixing, carries out extension;
(5) hybridization;
(6) the hybridization plate is carried out fluorescent scanning, judge the genotype of mononucleotide polymorphism site according to fluorescent signal.
2. a kind of method that detects 32 mononucleotide polymorphism sites simultaneously according to claim 1, it is characterized in that in the described step (2) the dilution of PCR primer and mix that to make its final concentration be 2.5 μ M, make up PCR primer pond, per 96 person-portion PCR reaction systems are:
Reagent becomes partial volume (μ L)
Primer pond (2.5 μ L) 11.2
Deoxynucleotide (2.5mM) 20
10 times of PCR damping fluid II 56.2
Magnesium chloride (25mM) 112.6
Gold medal amplification enzyme (5U/ μ L) 11.2
Distilled water 126
3. a kind of method that detects 32 mononucleotide polymorphism sites simultaneously according to claim 1 is characterized in that the PCR cycling condition is in the described step (2):
4. a kind of method that detects 32 mononucleotide polymorphism sites simultaneously according to claim 1 is characterized in that will extending in the described step (4) primer dilution and mixes that to make its final concentration be 5 μ M, makes up and extends the primer pond.Per 96 person-portion PCR reaction systems are:
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010100715A CN101851672A (en) | 2010-01-26 | 2010-01-26 | Method for simultaneously detecting 32 single nucleotide polymorphism (SNP) locus genotypes |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010100715A CN101851672A (en) | 2010-01-26 | 2010-01-26 | Method for simultaneously detecting 32 single nucleotide polymorphism (SNP) locus genotypes |
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| Publication Number | Publication Date |
|---|---|
| CN101851672A true CN101851672A (en) | 2010-10-06 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201010100715A Pending CN101851672A (en) | 2010-01-26 | 2010-01-26 | Method for simultaneously detecting 32 single nucleotide polymorphism (SNP) locus genotypes |
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| Country | Link |
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| CN (1) | CN101851672A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103122378A (en) * | 2012-12-20 | 2013-05-29 | 宁波大学 | Kit capable of being used for detecting methylation degree of alpha-adduction protein gene promoter region related to primary hypertension and application thereof |
| CN103122378B (en) * | 2012-12-20 | 2014-11-05 | 宁波大学 | Kit capable of being used for detecting methylation degree of alpha-adduction protein gene promoter region related to primary hypertension and application thereof |
| CN111455062A (en) * | 2020-04-01 | 2020-07-28 | 中国人民解放军总医院 | Kit and platform for detecting susceptibility genes of novel coronavirus |
| CN111455062B (en) * | 2020-04-01 | 2022-02-11 | 中国人民解放军总医院 | Kit and platform for detecting susceptibility genes of novel coronavirus |
| CN112195229A (en) * | 2020-09-07 | 2021-01-08 | 中国人民解放军火箭军特色医学中心 | Kit for simultaneously detecting multiple SNP sites related to radiosensitivity |
| CN112195229B (en) * | 2020-09-07 | 2022-07-12 | 中国人民解放军火箭军特色医学中心 | Kit for simultaneously detecting multiple SNP sites related to radiosensitivity |
| CN114164265A (en) * | 2021-11-10 | 2022-03-11 | 南通大学 | Group of genetic markers related to mycoplasma pneumonia of children and obtaining method thereof |
| CN114164265B (en) * | 2021-11-10 | 2024-04-16 | 南通大学 | Group of genetic markers related to mycoplasma pneumonia in children and obtaining method thereof |
| CN116179669A (en) * | 2023-01-04 | 2023-05-30 | 北京和合医学诊断技术股份有限公司 | Typing kit, primer and typing method for vitamin B12 metabolism related genes |
| CN116179669B (en) * | 2023-01-04 | 2023-10-24 | 北京和合医学诊断技术股份有限公司 | Typing kit, primer and typing method for vitamin B12 metabolism related genes |
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