CN100392101C - PPAR-alpha and PPAR-gamma gene polymorphism detecting chip preparing method and use - Google Patents
PPAR-alpha and PPAR-gamma gene polymorphism detecting chip preparing method and use Download PDFInfo
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- CN100392101C CN100392101C CNB200510061696XA CN200510061696A CN100392101C CN 100392101 C CN100392101 C CN 100392101C CN B200510061696X A CNB200510061696X A CN B200510061696XA CN 200510061696 A CN200510061696 A CN 200510061696A CN 100392101 C CN100392101 C CN 100392101C
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Abstract
The present invention discloses a preparing method and application of a chip for detecting the polymorphism of PPAR-alpha and PPAR-gamma genes. A probe which is shown in the right drawing is arranged on a carrier. The present invention has the advantage that the polymorphism of PPAR-alpha genes leu 162 val, val227 ala and a PPAR-gamma gene pro12 ala are detected and the characteristics of rapadity, simplity, directness and high flux. The present invention which is helpful to sieve the persons who are susceptible to obesity, diabetes, etc. to promote the development of gene diagnosis and therapies has significant social and economical benefits.
Description
Technical field
The present invention relates to the preparation method and the purposes of a kind of PPAR-α and PPAR-gamma gene polymorphism detecting chip.
Background technology
Peroxisome proliferation-activated receptors (peroxisome proliferator-activated receptor, PPARs) at first find when the nineteen ninety research mouse liver cDNA library by Isseman etc., be a class by part activated nuclear factor, belong to the member of nuclear receptor superfamily.The PPARs superfamily has three hypotypes: PPAR α, PPAR γ, PPAR β/δ, and by different genes encodings.Each hypotype of PPARs is at the expression degree varies sample of different tissues, and PPAR α mainly is distributed in the higher tissue of metabolic activity, as liver, kidney, heart, muscle tissue.PPAR γ mainly is expressed in fatty tissue and immunity system, and is in close relations with insulin resistant.
Yamakawa-Kobayashi K etc. discovers that the Val227Ala polymorphism of PPAR α is relevant with blood lipid level, and Ala227 carrier's total cholesterol level is starkly lower than non-Ala227 carrier [1].Kolehmainen etc. carry out PPARgamma2 gene Pro12Ala polymorphism analysis to 30 routine obese persons and find that the Ala12 gene frequency is 13.3%, body weight, fat thickness and empty stomach serum leptin level as broad as long [2] between each genotype.Gonzalez Sanchez JL finds that obese males Ala12 gene frequency (15%) is apparently higher than non-obese males (8%) [3].Patient's the leptin level (31.4ng/ml) that carries Pro12Ala sudden change is than not mutated person (17.5ng/ml) higher [4].
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, these relevant with the poisonous substance susceptibility with disease susceptibility [5].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 [6].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.
1.Yamakawa-Kobayashi?K，Ishiguro?H，Arinami?T，et?al.A?Val227Ala?polymorphism?inthe?peroxisome?proliferator?activated?receptor?alpha(PPARalpha)gene?isassociated?with?variations?in?serum?lipid?levels.J?Med?Genet.2002，39(3)：189-191.
2.Kolehmainen?M，Uusitupa?MI，Alhava?E，Laakso?M，Vidal?H.Effect?of?the?Pro12Alapolymorphism?in?the?peroxisome?proliferator-activated?receptor(PPAR)gamma2gene
3.on?the?expression?of?PPARgamma?target?genes?in?adipose?tissue?of?massively?obesesubjects.J?Clin?Endocrinol?Metab.2003Apr；88(4)：1717-1722.
4.Serrano?Rios?M，Fernandez?Perez?C，Laakso?M，Martinez，Larrad?MT.Effect?of?thePro12Ala?polymorphism?of?the?peroxisome?proliferator-activated?receptor?gamma-2gene?on?adiposity，insulin?sensitivity?and?lipid?profile?in?the?Spanish?population.Eur?J?Endocrinol.2002；147(4)：495-501.
5.Simon?I，Vendrell?J，Gutierrez?C，Fernandez-Real?JM，Vendrell?I，Gallart?L，FontovaR，Richart?C.Pro12Ala?substitution?in?the?peroxisome?proliferator-activatedreceptor-gamma?is?associated?with?increased?leptin?levels?in?women?with?type-2diabetes?mellitus.Horm?Res.2002；58(3)：143-149.
6.Khner?M?K，Beerli?P，Yamato?J，et?al.Usefulness?of?single?nucleatide?polymorphismdata?for?estimating?population?parameters.Genetics，2000，156：439-447
7.Hirsclhorn?J?N，Sklar?P，Lindblad-Toh?K，et?al.SBE-TAGS：an?array-based?method?forefficient?single-nucleotide?polymorphism?genotyping.Proc?Natl?Acad?SciUSA，2000，97：12164-12169
Summary of the invention
The preparation method and purposes PPAR-α and the PPAR-gamma gene polymorphism detecting chip that the purpose of this invention is to provide a kind of PPAR-α and PPAR-gamma gene polymorphism detecting chip: on carrier, be provided with following probe:
1 PPAR-α leu162val CAA?GTG?CAT?TTC?TGT?C
2 PPAR-α leu162val CAA?GTG?CGT?TTC?TGT?C
3 PPAR-α leu162val CAA?GTG?CCT?TTC?TGT?C
4 PPAR-α leu162val CAA?GTG?CTT?TTC?TGT?C
5 PPAR-α val227ala CCG?GGA?CAT?CCT?CT
6 PPAR-α val227ala CCG?GGG?CAT?CCT?CT
7 PPAR-α val227ala CCG?GGC?CAT?CCT?CT
8 PPAR-α val227ala CCG?GGT?CAT?CCT?CT
9 PPAR-γ pro12ala CTA?TTG?ACA?CAG?AAA?G
10 PPAR-γ pro12ala CTA?TTG?ACG?CAG?AAA?G
11 PPAR-γ pro12ala CTA?TTG?ACC?CAG?AAA?G
12 PPAR-γ pro12ala CTA?TTG?ACT?CAG?AAA?G
PPAR-α and PPAR-gamma gene polymorphism detecting chip preparation method step are:
1) processing of chip carrier: slide chromic acid lotion soaked overnight, washing, spend the night in 24～26% ammoniacal liquor, washing, slide immerses in 95% ethanolic soln of aminopropyl trimethoxysilane, regulates pH value 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 PPAR-α gene leu162val, val227ala polymorphism and PPAR-γ gene pro12ala polymorphism have designed one group of probe, its length range is in 14 ~ 16 bases (table 1), at PPAR-α gene leu162val, 3 pairs of primers have been synthesized in val227ala polymorphism and the design of PPAR-γ gene pro12ala polymorphism, its length range is in 21-22 base (table 2), a Cy3 signaling molecule mark is arranged in every primer, synthetic back 50～60 ℃ of deprotections of strong aqua, cut 13～17 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: with probe dilution to final concentration is 100 μ mol/L; With sampling liquid (750mmol/L sodium-chlor, the 75mmol/L sodium acetate, 5% glycerine, 1% ficoll and, 0.1% dodecyl sodium sulfonate) dilution in 1: 1, and get 10 μ L to 96 orifice plates, put to the aldehyde radical slide with the chip point sample instrument, every some volume is about 0.5nL, and the dessert spacing is 500 μ m, spot diameter is about 200 μ m, every probe repeats a little 2 times, and after point sample finished, room temperature was placed 24 hours immobilizations.
PPAR-α and PPAR-gamma gene polymorphism detecting chip are used to detect PPAR-α and PPAR-gamma gene polymorphism.
The present invention will be through the probe point sample selected on slide, hybridize respectively with through the PPAR-of pcr amplification α and PPAR-γ gene fragment, the polymorphism situation in each site of disposable acquisition, thus realize PPAR-α and PPAR-gamma gene polymorphism are carried out quick, simple and direct, high throughput testing and somatotype.Help susceptible persons' such as obesity, diabetes screening, can be used as the basis of prevention, diagnosis and the drug screening of disease, promote the development of gene diagnosis and treatment.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 the sample DNA extension increasing sequence 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.Method based on oligonucleotide chip provides a kind of high-throughout technology for analyzing SNP, for SNP or transgenation provide a high-throughout detection platform, can be used to sudden change or the SNP site of parallel analysis known in the various disease sample, change with the genetics that obtains diseases predisposing gene, and, have important society and economic benefit as clue reinforcement prevention early clinically, discovery early, diagnosis morning and early treatment.
Embodiment
Immobilised probe is an oligonucleotide probe (table 1) on the slide of the present invention, and its sequence is according to PPAR-α gene leu162val, val227ala polymorphism and PPAR-γ gene pro12ala polymorphism characteristic Design.Said primer is at PPAR-α and PPAR-gamma gene polymorphism characteristic Design synthetic primer (table 2), is used for the amplification of said gene.
The signaling molecule mark is the primer at PPAR-α and the amplification of PPAR-gamma gene sequences.The signaling molecule mark is a fluorescence molecule.
The probe of table 1:PPAR-α and PPAR-gamma gene polymorphism detecting chip
1 PPAR-α leu162val CAA?GTG?CAT?TTC?TGT?C
2 PPAR-α leu162val CAA?GTG?CGT?TTC?TGT?C
3 PPAR-α leu162val CAA?GTG?CCT?TTC?TGT?C
4 PPAR-α leu162val CAA?GTG?CTT?TTC?TGT?C
5 PPAR-α val227ala CCG?GGA?CAT?CCT?CT
6 PPAR-α val227ala CCG?GGG?CAT?CCT?CT
7 PPAR-α val227ala CCG?GGC?CAT?CCT?CT
8 PPAR-α val227ala CCG?GGT?CAT?CCT?CT
9 PPAR-γ pro12ala CTA?TTG?ACA?CAG?AAA?G
10 PPAR-γ pro12ala CTA?TTG?ACG?CAG?AAA?G
11 PPAR-γ pro12ala CTA?TTG?ACC?CAG?AAA?G
12 PPAR-γ pro12ala CTA?TTG?ACT?CAG?AAA?G
The primer of table 2:PPAR-α and PPAR-gamma gene polymorphism detecting chip
Gene forward primer reverse primer (or fluorescent mark reverse primer)
PPAR-α 5’-CTC?AAG?CTG?GTG?TAT?GAC?AAG 5’-CGA?ACT?GGG?AAA?ATG?TGC?AGG-3’
T-3’
PPAR-α 5’-CTC?TGG?CCA?AGA?GAA?TCT?ACG 5’-GTT?CCA?TGT?TGC?CAA?GAG?AAC?C
-3’ -3’
PPAR-γ 5’-GGA?TAT?TGA?ACA?GTC?TCT?GCT 5’-GTT?TGC?AGA?CAG?TGT?ATC?AGT?G
C-3’ -3’
Embodiment 1: the preparation of nucleotide gene chip
1) processing of chip carrier: slide chromic acid lotion soaked overnight, washing is spent the night in 25% ammoniacal liquor, washing.Slide immerses in 95% ethanolic soln of aminopropyl trimethoxysilane, regulates pH value to 4.5 with Glacial acetic acid, and 95% ethanol ultrasonic cleaning was dried 4 hours for 160 ℃, and 10% glutaraldehyde is processed into aldehyde radicalization.
2) primer and probe is synthetic: designed one group of probe (seeing Table 1) at PPAR-α gene leu162val, val227ala polymorphism and PPAR-γ gene pro12ala pleomorphism site.6 primers (seeing Table 2) have been synthesized at PPAR-α gene leu162val, val227ala polymorphism and the design of PPAR-γ gene pro12ala pleomorphism site.A Cy3 fluorescent mark is arranged in every primer.Synthetic back was cut the OPC column purification 15 hours with 55 ℃ 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: with probe dilution to final concentration is 100 μ mol/L, with sampling liquid (750mmol/L sodium-chlor, the 75mmol/L sodium acetate, 5% glycerine, 1% ficoll and, 0.1% dodecyl sodium sulfonate) dilution in 1: 1, and get 10 μ L to 96 orifice plates, put to the aldehyde radical slide with chip point sample instrument (Cartisan), every some volume is about 0.5nL, the dessert spacing is 500 μ m, and spot diameter is about 200 μ m, and every probe repeats a little 2 times, after point sample finished, room temperature was placed 24 hours immobilizations.
Embodiment 2: 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 24% ammoniacal liquor, washing.Sheet glass immerses in 95% ethanolic soln of aminopropyl trimethoxysilane, regulates pH value to 4.0 with Glacial acetic acid, and 95% ethanol ultrasonic cleaning was dried 4 hours for 150 ℃, and 9% glutaraldehyde is processed into aldehyde radicalization.
2) primer and probe is synthetic: designed one group of probe, (seeing Table 1) at PPAR-α gene leu162val, val227ala polymorphism and PPAR-γ gene pro12ala pleomorphism site.6 primers (seeing Table 2) have been synthesized at PPAR-α gene leu162val, val227ala polymorphism and the design of PPAR-γ gene pro12ala pleomorphism site.A Cy3 fluorescent mark is arranged in every primer.Synthetic back was cut the OPC column purification 13 hours with 50 ℃ 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: with probe dilution to final concentration is 100 μ mol/L, with sampling liquid (750mmol/L sodium-chlor, the 75mmol/L sodium acetate, 5% glycerine 1,1% ficoll and, 0.1% dodecyl sodium sulfonate) dilution in 1: 1, and get 10 μ L to 96 orifice plates, put to the aldehyde radical slide with chip point sample instrument (Cartisan), every some volume is about 0.5nL, the dessert spacing is 500 μ m, and spot diameter is about 200 μ m, and every probe repeats a little 2 times, after point sample finished, room temperature was placed 24 hours immobilizations.
The preparation of embodiment 3 nucleotide gene chips
1) processing of chip carrier: slide chromic acid lotion soaked overnight, washing is spent the night in 26% ammoniacal liquor, washing.Sheet glass immerses in 95% ethanolic soln of aminopropyl trimethoxysilane, regulates pH value to 4.2 with Glacial acetic acid, and 95% ethanol ultrasonic cleaning was dried 5 hours for 155 ℃, and 11% glutaraldehyde is processed into aldehyde radicalization.
2) primer and probe is synthetic: designed one group of probe, (seeing Table 1) at PPAR-α gene leu162val, val227ala polymorphism and PPAR-γ gene pro12ala pleomorphism site.6 primers (seeing Table 2) have been synthesized at PPAR-α gene leu162val, val227ala polymorphism and the design of PPAR-γ gene pro12ala pleomorphism site.A Cy3 fluorescent mark is arranged in every primer.Synthetic back was cut the OPC column purification 17 hours with 60 ℃ 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: with probe dilution to final concentration is 100 μ mol/L, with sampling liquid (750mmol/L sodium-chlor, the 75mmol/L sodium acetate, 5% glycerine, 1% ficoll and, 0.1% dodecyl sodium sulfonate) dilution in 1: 1, and get 10 μ L to 96 orifice plates, put to the aldehyde radical slide with chip point sample instrument (Cartisan), every some volume is about 0.5nL, the dessert spacing is 500 μ m, and spot diameter is about 200 μ m, and every probe repeats a little 2 times, after point sample finished, room temperature was placed 24 hours immobilizations.
The using method of PPAR-α and PPAR-gamma gene polymorphism is:
1) respectively washes twice with 0.2%SDS and clear water, 1%NaBH after the dry air before the slide that is loaded with oligonucleotide probe uses
4Solution reduction 10min, 0.2%SDS wash once, and washing once with stand-by, is finished probe covalency immobilization in surface of glass slide after the dry air.
2) method that adopts asymmetric PCR to increase for the target fragment that produces strand, the ratio optimization of forward primer and reverse primer (or fluorescent mark reverse primer) is 1: 10.Contain 1.5mMMgCl in the 20 μ L reaction systems
2, each 200 μ mol/L of dNTP, forward primer 0.1 μ mol, reverse primer 1 μ mol, DNA 50ng, 1 * PCR damping fluid and 1U Taq enzyme.The pcr amplification condition is: pre-94 ℃ of 5min of sex change; 94 ℃ of 30sec of sex change, the 58 ℃ of 30sec that anneal extend 72 ℃ of 30sec, totally 30 circulations; Extend 72 ℃ of 5min at last.The PCR product is with 2% agarose gel electrophoresis analysis.
3) the asymmetric PCR product of Cy3 mark and hybridization solution (750mmol/L NACl, 75mmol/L sodium acetate, 0.1%SDS, 1ug/ml salmon sperm DNA, 2.5% formyl ammonium) mixed by 2: 8,10 μ L mixed solutions were transferred to the hybridization zone of chip.Chip places hybridizing box 42 ℃ of water-bath insulations 1 hour.Chip is taken out in the hybridization back, and (150mmol/LNACl, 15mmol/L sodium acetate 0.2%SDS), respectively washed 1 minute among washing lotion B (30mmol/L NACl, 3mmol/L sodium acetate) and the washing lotion C (15mmol/L NACl, 1.5mmol/L sodium acetate) at washing lotion A successively.Wait to do the back chip and use laser confocal scanning instrument GenePix4000 (Axon Instrument) at excitation wavelength 532nm, emission wavelength 570nm (Cy3) scanning produces and analysis precision is 16 TIFF images of 10 μ m.
PPAR-α and PPAR-gamma gene polymorphism detecting chip use-case:
Accept health check-up 180 routine grownups in Zhejiang University Medical College The First Affiliated Hospital,, be divided into 2 groups: (1) fatty liver group 117 examples, male 77 examples, women 40 examples, 47.97 ± 13.46 years old age according to the Case definition of fatty liver; (2) control group 63 examples, male 32 examples, women 31 examples, 55.13 ± 10.92 years old age.Measure height, body weight, hip circumference, waistline, stomach wall fat thickness, body fat content, blood pressure simultaneously.All selected objects are all signed Informed Consent Form.Subjects is carried out the empty stomach blood sampling, and the part blood specimen is used to detect total protein, albumin, propyl group propylhomoserin transferring enzyme, triglyceride level, high-density lipoprotein (HDL) and glucose level.The part blood specimen extracts genomic dna with Genomic DNA purification kit (Promega company), and behind pcr amplification, PCR product and the oligonucleotide chip hybridization, washing and the scanning that prepare detect PPAR-α and PPAR-gamma gene polymorphism.The result is: 162 Nucleotide of (1) PPAR-α gene are wild-type, do not find PPAR-α gene leu162val sudden change.(2) the 227th Nucleotide CC of PPAR-α gene (Ala227), CT (Val227Ala) and TT (Val227) genotype are respectively in 63 routine normal populations and are 1,12 and 50 examples, and in 117 routine non-alcohol fatty liver patients, CC (Ala227), CT (Val227Ala) and TT (Val227) genotype are respectively 2,7 and 108 examples, and the distribution frequency of allele C and T has significance difference (P≤0.01) between two groups.(3) CC (pro12) and GC (pro12ala) genotype are respectively 57 example and 6 examples in the 227th Nucleotide pro12ala63 example of the PPAR-γ gene normal population, and 117 routine non-alcohol fatty liver patient CC (pro12) and GC (pro12ala) genotype are respectively 104 example and 13 examples.
PCR product sequence verification
The order-checking of PCR product is according to CEQ
TMTDCS test kit (BECKMAN COULTER
TM, USA) specification sheets operation is at CEQ
TM2000XL sequenator (BECKMAN COULTER
TM, carry out on USA).Sequencing result is consistent with the oligonucleotide microarray technique detected result.
Claims (4)
1. PPAR-α and PPAR-gamma gene polymorphism detecting chip is characterized in that: be provided with following probe on carrier:
1?PPAR-α leu162val CAA?GTG?CAT?TTC?TGT?C
2?PPAR-α leu162val CAA?GTG?CGT?TTC?TGT?C
3?PPAR-α leu162val CAA?GTG?CCT?TTC?TGT?C
4?PPAR-α leu162val CAA?GTG?CTT?TTC?TGT?C
5?PPAR-α val227ala CCG?GGA?CAT?CCT?CT
6?PPAR-α val227ala CCG?GGG?CAT?CCT?CT
7?PPAR-α val227ala CCG?GGC?CAT?CCT?CT
8?PPAR-α val227ala CCG?GGT?CAT?CCT?CT
9?PPAR-γ pro12ala CTA?TTG?ACA?CAG?AAA?G
10?PPAR-γ?pro12ala CTA?TTG?ACG?CAG?AAA?G
11?PPAR-γ?pro12ala CTA?TTG?ACC?CAG?AAA?G
12?PPAR-γ?pro12ala CTA?TTG?ACT?CAG?AAA?G
2. a kind of PPAR-α according to claim 1 and PPAR-gamma gene polymorphism detecting chip is characterized in that: said carrier is a slide.
3. the preparation method of PPAR-α and PPAR-gamma gene polymorphism detecting chip, it is characterized in that: method steps is:
1) processing of chip carrier: slide chromic acid lotion soaked overnight, washing, spend the night in 24～26% ammoniacal liquor, washing, slide immerses in 95% ethanolic soln of aminopropyl trimethoxysilane, regulates pH value 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: designed one group of probe at PPAR-α gene leu162val, val227ala polymorphism and PPAR-γ gene pro12ala pleomorphism site, its length range is 14～16 bases, at 2 pairs of primers of PPAR-α gene design, at the synthetic 1 pair of primer of PPAR-γ gene design, downstream primer Cy3 fluorescent mark, synthetic back 50～60 ℃ of deprotections of strong aqua, cut 13～17 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: with probe dilution to final concentration is 100 μ mol/L, with sampling liquid dilution in 1: 1, the component of sampling liquid is a 750mmol/L sodium-chlor, the 75mmol/L sodium acetate, 5% glycerine, 1% ficoll and, 0.1% dodecyl sodium sulfonate, and get 10 μ L to 96 orifice plates, put to the aldehyde radical slide with the chip point sample instrument, every some volume is 0.5nL, and the dessert spacing is 500 μ m, spot diameter is 200 μ m, every probe repeats a little 2 times, and after point sample finished, room temperature was placed and got final product in 24 hours.
Said probe is:
1?PPAR-α leu162val CAA?GTG?CAT?TTC?TGT?C
2?PPAR-α leu162val CAA?GTG?CGT?TTC?TGT?C
3?PPAR-α leu162val CAA?GTG?CCT?TTC?TGT?C
4?PPAR-α leu162val CAA?GTG?CTT?TTC?TGT?C
5?PPAR-α val227ala CCG?GGA?CAT?CCT?CT
6?PPAR-α val227ala CCG?GGG?CAT?CCT?CT
7?PPAR-α val227ala CCG?GGC?CAT?CCT?CT
8?PPAR-α val227ala CCG?GGT?CAT?CCT?CT
9?PPAR-γ pro12ala CTA?TTG?ACA?CAG?AAA?G
10PPAR-γ pro12ala CTA?TTG?ACG?CAG?AAA?G
11PPAR-γ pro12ala CTA?TTG?ACC?CAG?AAA?G
12PPAR-γ pro12ala CTA?TTG?ACT?CAG?AAA?G
4. the preparation method of a kind of PPAR-α according to claim 3 and PPAR-gamma gene polymorphism detecting chip, it is characterized in that: said primer is according to PPAR-α and the design of PPAR-γ gene expression characteristics, be respectively applied for the amplification of said gene, said PPAR-α primer 1 is: forward primer (5 '-CTC AAG CTG GTG TAT GAC AAG T-3 ') and reverse primer (5 '-CGA ACT GGG AAA ATG TGC AGG-3 '); Said PPAR-α primer 2 is: forward primer (5 '-CTC TGG CCA AGA GAA TCT ACG-3 ') and reverse primer (5 '-GTT CCA TGT TGC CAA GAG AAC C-3 '); Said PPAR-γ gene primer is: forward primer (5 '-GGA TAT TGA ACA GTC TCT GCT C-3 ') and reverse primer (5 '-GTT TGC AGA CAG TGT ATC AGT G-3 ').
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