CN100368564C - Primer, method and kit for identifying PGC-1 gene promotor 1998 point SNP molecular mark - Google Patents

Abstract

The present invention discloses a method for finding and typing the 1998th SNP molecular label of a PGC-1 gene promoter in north crowd of China. The method of a PCR is introduced by extracting the genome DNA of host cells, according to the specific design primer in the present invention, and by adopting nest type PCR combined and restrictive sites. The method measures the genotype of the 1998th SNP label of the PGC-1 gene promoter of a subject, and combines SNP labels existing in other PGC-1 sequences to predict the susceptibility of the subject to a metabolic syndrome. A typing result of the method is consistent with that of a method for controlling the quality and measuring sequence of ribonucleotide, and the typing result is reliable. The present invention has the advantages that the 1998th C/G SNP label of the PGC-1 gene promoter existing in the north crowd of China is found for the first time. The method for selecting and typing the SNP label, and specific primers are provided. After building a monomer type, the method is directly applied to the north crowd in China to develop the positioning and the selection of pathogenic genes of the metabolic syndrome, and the method can be applied to the evaluation of the individual pathogenic risk of the metabolic syndrome. Thus, the present invention is favorable to develop the early intervention and the cure of the metabolic syndrome.

Description

A kind of primer, method and test kit of identifying the PGC-1 gene promotor 1998 point SNP molecular mark

Technical field

The present invention relates to primer, method and the test kit of-1998 SNP molecule markers in a kind of PGC-1 of evaluation gene promoter sequence, belong to biological technical field.

Background technology

Single nucleotide polymorphism SNP belongs to third generation molecule marker, and after human genome DNA's sequence order-checking and analytical work were finished, it can provide strong application to provide safeguard for systematic research gene function and correlated inheritance disease.Because it is distributed widely in human genome and relatively stable existence, thereby in using, the hereditary mechanism research of disease related gene location and disease has prospect.By the SNP mark of finding to exist in the special group, and the design different schemes carries out the crowd size with the SNP that finds and divides the type examination, provides technical foundation and means with the direct mechanism research for heredopathia; Simultaneously, etiological diagnosis, treatment and the control to the human genetic disease produces great effect.

Discover that the mean density of SNP in human genome is estimated as 1/1000bp, reach 3 * 10 in the distribution of whole genome ⁶Individual, and density is higher than s-generation molecule marker STR.Most of SNP derive from after the species formation, before population occurs, because the variation frequency of Nucleotide is extremely low by (about 10 in each generation ^-8) and the randomness that changes of base, making that single base allelotrope is very stable, most of (85%) is (but the frequency difference that takes place in different population) that has among the human SNP.Find the SNP that exists in the special group, and study its regularity of distribution will be for thorough relatively and disclose comprehensively that disease gene location situation provide strong point of penetration in the human genome.

Though finishing of human genome DNA's sequence examining order has and great Practical significance the mankind, particularly in the field of etiologic etiological searching of disease genetic and population genetics; But, because finishing of the Human Genome Project be based on the finite population basis, during at special group (as intercontinental, national, area etc.) research, then need be under the prerequisite of reference and reference the concrete analysis problem.Briefly; choosing specific human genome section according to concrete research Application Areas exactly analyzes in conjunction with human genome DNA's sequence results in the special group object; the constitutional features that exists in its genome section in the screening special group object (being molecule marker) with biological significance; and the molecule marker that physical presence in the special group can be studied carried out mass-producing, high-throughout somatotype, thereby establish technical foundation for follow-up wide Application Areas.

Relate to peroxisome proliferation-activated receptors γ co-activator 1 gene (peroxisomeproliferator-activated receptor-γ coactivator-1, PGC-1) discovery of SNP molecule marker in the northern colony of China and the example of mass-producing parting kit in the promoter sequence among the present invention.PGC-1 is a kind of multi-functional transcription regulaton factor, can participate in regulating all many-sides of energy metabolism, stimulates plastosome biosynthesizing and plastosome oxidative metabolism, to adapt to different metabolic demand.Its expression is subjected to cold, hunger, endurance exercise, infection etc. to stimulate regulation and control, to regulate metabolism stable states such as plastosome energy metabolism, glycogen heteroplasia, adapts to the body needs.It can wide participation plastosome biosynthesizing and the characteristics of the adjusting of many metabolic pathways such as energy metabolism, glycogen metabolism and lipid acid beta-oxidation, has indicated that the PGC-1 gene may have application prospect in the fundamental research of metabolism syndrome and clinical intervention.

In the eukaryotic gene expression regulation and control, transcriptional control is the link of most critical, and transcription factor combines by the promotor with the target gene, thereby promotes or the inhibition target gene expression, changes the biology program, finally influences the organism vital movement.Carry out examination by the promoter region to the target gene, the SNP mark in the specific crowd that can find may exist in its promotor nucleotide sequence is realized the Position Research of Disease-causing gene indirectly by linkage analysis and linkage disequilibrium analysis on the one hand; Also may directly find the pathogenic mutation SNP in the promotor on the other hand, because this SNP undergos mutation, changed transcription factor and be arranged in the bonding state of the cis-regulating element of promotor, changed, finally caused the generation of pathological state thereby influence the target gene expression amount.As seen, in the research that disease genetic is learned, the scanning of gene promoter section is the powerful measure of finding SNP characteristic distributions and rule in the special group.

Through existing domestic and foreign literature retrieval, the whole world is not seen any about the identification of third generation molecular marker SNP and the report of somatotype research method in the human PGC-1 gene promoter region sequence, does not see SNP and/or the haplotype of its participation structure and the relevant report of metabolism syndrome research in any human PGC-1 gene promoter yet.

Summary of the invention

The purpose of this invention is to provide a kind of specificity method design and PCR primer sequence of finding and identifying Chinese northerner PGC-1 gene promoter region-1998 a SNP mark.

Second purpose of the present invention provides a kind of easy to use, highly sensitive detection kit that comprises above-mentioned primer.

For achieving the above object, the present invention is by the following technical solutions:

SNP molecular marker method in a kind of evaluation PGC-1 gene promoter sequence, by extracting the genomic dna of host cell, adopt the method for nest-type PRC, measure experimenter's PGC-1 gene promotor 1998 point SNP marker genotypes in conjunction with restriction site introducing PCR; Wherein the outer primer sequence of nest-type PRC such as sequence table SEQ ID No.4 and SEQ ID No.5; Inner primer is primer sequence such as sequence table SEQ ID No.6 and the SEQ ID No.7 that restriction site is introduced PCR.

A kind of isolating nucleic acid has the base sequence shown in the sequence table SEQ ID NO.1, is the SNP mark of C/G multiple allelomorphos (representing with  in the SEQ ID NO.1 sequence) at the-1998.Naming method is: in PGC-1 gene translation initiator codon ATG, when VITAMIN B4 A was defined as No. 1 position, thymus pyrimidine T, guanine G were respectively the position 2, No. 3 in its downstream sequence; In like manner, its upstream sequence is followed successively by 0 ,-1 ,-2 ... the position, in the northern crowd of China, the-1998 is the C/G polymorphism mark.Nucleotides sequence shown in the SEQ ID NO.1 is classified part PGC-1 gene promoter sequence as.This genome sequence can obtain from Genbank, original gene group complete sequence being numbered in Genbank (AC092834).

Those skilled in the art is clear, has a lot of analytical procedures can be used for detecting the PGC-1 gene promoter sequence and whether has single nucleotide polymorphism.These technology comprise (but being not limited to): dna sequencing, sequencing by hybridization, DNA chip, PCR-RFLP, PCR-SSCP, enzymatic mispairing cutting, denaturing gradient gel electrophoresis, sex change high performance liquid chromatography (DHPLC) etc.

First by direct sequencing, in the northern crowd of China between 30 diabetes B patients and 10 ages, gender matched normal control colony among the Chinese northern crowd of discovery PGC-1 promoter region-1998 have C/G polymorphism SNP marker site.Be used for the inventive method or detection kit primer based on special design, (Restriction sitegeneration-PCR RG-PCR) realizes PGC-1 promoter region-1998 a C/G polymorphism SNP marker site genotyping to adopt two-step approach nest-type PRC (nested-PCR) engagement limits site to generate the PCR method.Carry out obtaining sequence fragment shown in the SEQ ID NO.2 after the nido the first step PCR reaction amplification, employed primer has following sequence (SEQ ID NO.4, SEQ ID NO.5):

SEQ?ID?NO.4?5’-GGG?CAG?GTT?AGA?ATA?TCA?A-3’；

SEQ?ID?NO.5?5’-GGC?TAA?TGC?AGG?TAG?GTG-3’。

Generate the designed primer of PCR method in conjunction with restriction site, being used to carry out the PCR reaction of second step of nido uses, after the digestion with restriction enzyme reaction, can specificity identify this SNP mark, obtain sequence fragment shown in the SEQ IDNO.3 after the amplification, employed primer has following sequence (SEQ ID NO.6, SEQ ID NO.7):

SEQ?ID?NO.6?5’-CAG?TAG?TAT?TTC?ACA?GTG?GAT?C-3’；

SEQ?ID?NO.7?5’-AAT?AAC?AGG?AAA?ATA?CAA?CCT?C-3’。

A kind of easy to use, highly sensitive detection kit that comprises above-mentioned primer, it contains described PCR specific amplification primer (SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7) and is used for the conventional assembly of the test kit of pcr amplification detection, reagent, damping fluid etc., and those skilled in the art know these conventional assembly and detection methods.Detection amplified production and normal the-1998 SNP of PGC-1 gene promoter area contrast when whether having variation mutually, and required chemical reagent also comprises specific restriction enzyme etc.Whole components, content, source and using method in the test kit of the present invention are as follows:

Test kit of the present invention detects for 10 person-portions and uses, and its component, content and source comprise:

50 μ L, 10 * PCR damping fluids (Pharmacia),

50 μ L 10mM dNTP mixed solutions (Pharmacia),

10 μ L (2unit/ μ L) Taq archaeal dna polymerase (TAKARA),

Each 5 μ L (10pmol/ μ L) SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 primer,

The 1ml pure water,

20 μ L, 10 * K restriction enzyme reaction damping fluids (TAKARA),

5 μ L (10u/ μ L) restriction enzyme BamH I (TAKARA).

Using method:

1) by pcr amplification PGC-1 Gene Partial promoter fragment, in brief, the preparation mixed solution, add genomic dna solution 20ng, 2 μ L, 10 * PCR damping fluid, 0.4 μ L 10mM dNTP, 0.5 μ L Taq archaeal dna polymerase, 0.3 μ L sense primer (SEQ ID NO.4,10pmol/ μ L), 0.3 μ L antisense primer (SEQ ID NO.5,10pmol/ μ L), 15.5 μ L pure water, making cumulative volume is 20 μ L.The PCR reaction conditions is 95 ℃ of pre-sex change of 5mi n, 94 ℃ of 45s, 57 ℃ of 45s, 72 ℃ of 60s, 25 circulations, and 72 ℃ are extended 7min.After reaction is finished,, be the fragment that 1055bp, obtained single band, be and increase successfully as amplification with 3 μ L PCR reaction solutions electrophoresis detection on 8.0% polyacrylamide gel.

2), on first time pcr amplification basis, carry out nest-type PRC amplification PGC-1 Gene Partial promoter fragment by after the base mismatch design.Polyacrylamide gel electrophoresis is detected the successful PCR product 2 μ L first time of amplification to add in the 18 μ L pure water, therefrom take out 1 μ L behind the mixing, add 2 μ L, 10 * PCR damping fluid, 0.4 μ L 10mM dNTP, 0.5 μ L Taq archaeal dna polymerase, 0.3 μ L sense primer (SEQ ID NO.6,10pmol/ μ L), 0.3 μ L antisense primer (SEQ ID NO.7,10pmol/ μ L), 15.5 μ L pure water, making cumulative volume is 20 μ L.The PCR reaction conditions is 95 ℃ of pre-sex change of 3min, 94 ℃ of 45s, 58 ℃ of 45s, 72 ℃ of 45s, 30 circulations, and 72 ℃ are extended 7min.After reaction is finished,, be the fragment that 193bp, obtained single band, be and increase successfully as amplification with 3 μ L PCR reaction solutions electrophoresis detection on 8.0% polyacrylamide gel.

3) measure PGC-1 gene promoter region-1998 a SNP polymorphism by handle the PCR reaction product with restriction enzyme.Detect amplification from polyacrylamide gel electrophoresis and draw 3 μ L the PCR product the successful second time, add 1 μ L10 * K restriction enzyme reaction damping fluid, 0.3 μ L restriction enzyme BamH I (10u/ μ L), pure water 5.7 μ L, in 37 ℃ of water-baths, digest 4h behind the mixing.Afterwards, on 8.0% polyacrylamide gel, carry out electrophoresis.

4) polymorphism typing: handle fragment with restriction enzyme BamH I, when PGC-1 gene promoter region-1998 a SNP base is C allelotrope, the band of 175bp and 18bp occurs.As when being G allelotrope, band of 193bp only appears.

Measuring method of the present invention has been measured the genomic dna that derives from the people, sample without limits as, body fluid (as blood, ascites and urine), histocyte (as hepatic tissue) etc.Can prepare genomic dna by extraction and these samples of purifying.

From genomic dna, can increase contains the nucleic acid fragment of PGC-1 gene promotor 1998 point catastrophe point, with the great amount of samples that obtains to measure.Thisly generate the sample that the PCR method design obtains containing the dna fragmentation acquisition of PGC-1 transgenation point, be suitable for use as the mensuration material, carry out identification and the somatotype of follow-up SNP by two-step approach nest-type PRC engagement limits site.

Unrestricted to this design, as long as different in two kinds of situations of mutator gene and wild type gene with the fragment length of enzyme cutting DNA district acquisition.Therefore, according to the required dna fragmentation of PCR method amplification, the restriction enzyme by above-mentioned choose reasonable is designed.The fragment that enzyme is cut generation confirms that by electrophoresis they show specific band.According to the banding pattern that in aforesaid method, obtains, can measure the polymorphism of PGC-1 gene promoter region-1998 SNP.

When utilizing the present invention to carry out allelic gene typing, preferably be used in the auxiliary diagnostic of mensuration according to the mutation type existence of PGC-1 gene, auxiliary diagnostic comprises the particular agent as neccessary composition, and it is corresponding to the method that is used to measure the PGC-1 gene mutation type.Specific reagent is suitably selected by the measuring method that adopts.

Advantage of the present invention is: the present invention has found first among the Chinese northern crowd that there is SNP molecule marker (C/G) in PGC-1 gene promoter region-1998, and provide a kind of easy to use, highly sensitive detection reagent cassette method that comprises above-mentioned primer in detail, this method can be used for identification and the evaluation of PGC-1 promoter region-1998 SNP, can be used for comprising the early stage auxiliary diagnosis and the examination of disease such as metabolism syndrome in the Chinese northern colony then.

The present invention will be further described below in conjunction with embodiment, so that the public is to having more detailed understanding in the present invention, be not limitation of the invention, everyly any well known in the artly be equal to replacement, all belong to infringement of the present invention according to what the disclosure of invention was carried out.

Description of drawings

Fig. 1 is polynucleotide sequence result identification PGC-1 gene promoter-1998 (C/G) SNP mark after the information biology comparison of PCR-direct sequencing gained.

Fig. 2 is a BamH I restriction enzyme digestion and electrophoresis collection of illustrative plates.Wherein, 1,5,6,7,8,11,12 swimming lanes are the G/G genotype; 2,3,4,9 swimming lanes are the C/G genotype, and 10 swimming lanes are the C/C genotype, and 13 swimming lanes are blank, and 14 swimming lanes are molecular weight marker (SD011 of ancient cooking vessel state company).

Fig. 3 is that BamH I test kit restriction enzyme digestion and electrophoresis classifying method result is by the dna sequencing proof diagram.PGC-1 promotor-1998 (C/G) SNP uses this patent test kit genotyping result consistent with the dna sequencing result.

Embodiment

The english abbreviation that is used for the following example expression reagent is as follows.

EDTA: disodium ethylene diamine tetraacetate

SDS: sodium lauryl sulphate

TE：10mM?Tris-HCl(pH7.5)，1mM?EDTA(pH8.0)

10 * PCR damping fluid: 100mM Tris-HCl (pH8.3), 500mM KCl, 15mM magnesium chloride (MgCl2) 0.01% (w/V) gelatinum

DNTP: deoxynucleoside triphosphate

APS: ammonium persulphate → 10 * TBE:Tris (108g), boric acid (55g), EDTA2Na (9.3g) is dissolved in the pure water, and cumulative volume is 1 liter.

The extraction of collection of embodiment 1 blood sample and genomic dna

By WHO (1999) the standard MethodsThe cases enrolled of clarifying a diagnosis, must be through empty stomach 8-12 hour the oral 75g dextrose anhydrous that dissolves in the 300ml warm water, the diabetic subject who detects its plasma glucose value 〉=11.1mmol/L or clarified a diagnosis via the above rank in counties and cities hospital in the past after 2 hours, collect altogether from Heilungkiang and the affinity-less relation T2DM patient of Pekinese 396 examples, 50.0 years old ± 12.7 years old mean age, the male sex's 191 examples wherein, with geographic normal healthy controls volunteer's 319 examples, 45.2 years old ± 5.7 years old mean age, the wherein male sex's 181 examples.All persons under inspection are Han nationality, and the signature Informed Consent Form, and this research has also obtained the approval of Ethics Committee of our unit.

According to following method, prepare genomic dna with human peripheral.In the presence of antithrombotics EDTA, at 2500rpm, centrifugation removed serum deprivation in 30 minutes with the 10ml human peripheral collected.Then add 0.2%NaCl solution, making cumulative volume is 50ml.Vibrate gently solution 5-6 time, and it was positioned over 15 minutes on ice.After this, at 2500rpm centrifugation 30 minutes, collecting precipitation thing whereby.NaCl solution with 0.2% washs in the mode that is similar to the front again.In the throw out that so obtains, add 10mMTris-HCl (pH8.0) and 10mM EDTA (4ml), with this throw out that suspends.With 10%SDS, the Proteinase K of 25mg/ml and the RNaseA of 10mg/ml add in the suspension, and its add-on is respectively 4ml, 16ul and 20ul, and the suspension that then turns upside down mixes gently.Then, at 37 ℃ of incubation suspensions that spend the night.After spending the night, add 4ml phenol/Tris solution, the mixture of the mixture mixing gained that turns upside down.Carry out centrifugation with 3000rpm and removed water layer in 10 minutes.Water layer and 4ml phenol/chloroformic solution are mixed, then against mixing and with 3000rpm centrifugation 10 minutes.Remove water layer.At last, with chloroform extraction twice, obtaining water, toward wherein adding 1/10,3M NaAC (pH5.2), the cold dehydrated alcohol of doubling dose makes the DNA precipitation.Washing with alcohol with 70% is to obtain genomic dna.Make the DNA of such acquisition, genomic dna is dissolved among the TE, and the quantitative assay mixture is in the absorbancy of 260nm then.DNA working fluid concentration correction is put-20 ℃ of refrigerators and is preserved to 50ng/ul.

The identification of embodiment 2SNP is determined

The present invention adopts two-step approach nest-type PRC, base mismatch introducing restriction site method the PGC-1 gene promoter region to be carried out the examination of SNP mark and carry out the somatotype evaluation.

1) Auele Specific Primer is as follows:

SEQ?ID?NO.4?5’-GGG?CAG?GTT?AGA?ATA?TCA?A-3’；

SEQ?ID?NO.5?5’-GGC?TAA?TGC?AGG?TAG?GTG-3’。

SEQ?ID?NO.6?5’-CAG?TAG?TAT?TTC?ACA?GTG?GAT?C-3’；

SEQ?ID?NO.7?5’-AAT?AAC?AGG?AAA?ATA?CAA?CCT?C-3’

2) pcr amplification PGC-1 Gene Partial promoter fragment: the genomic dna solution 20ng, 2 μ L, 10 * PCR damping fluid, 0.4 μ L 10mM dNTP, 0.5 μ L Taq archaeal dna polymerase, 0.3 μ L sense primer (the SEQ ID NO.4 that add embodiment 1 preparation in the reaction vessel, 10pmol/ μ L), 0.3 μ L antisense primer (SEQ ID NO.5,10pmol/ μ L), 15.5 μ L pure water, making cumulative volume is 20 μ L.The PCR reaction conditions is 95 ℃ of pre-sex change of 5mi n, 94 ℃ of 45s, 57 ℃ of 45s, 72 ℃ of 60s, 25 circulations, and 72 ℃ are extended 7mi n.After reaction was finished, with 3 μ L PCR reaction solutions electrophoresis on 8.0% polyacrylamide gel, Bio-Rad gel imaging system Gel Doc2000 detected, and is the fragment that 1055bp as amplification, has obtained single band, is and increases successfully.

3) fragment of amplification gained 1055bp is directly carried out the order-checking of PCR product behind the purifying.Entrust China big-and-middle living development in science and technology company limited to carry out sequencing work.Originally be operated among the Chinese northern crowd in 30 diabetes B patients and 10 ages, the gender matched normal control colony and carry out.Sequencing result is (see figure 1) after GENESTAR instrument information biology multisequencing comparison, finds in the Chinese northern colony that there is the polymorphic marker site of C/G in PGC-1 promoter region-1998.

4) base mismatch design inner primer carries out the secondary PCR amplification: after biological letter opens, learns design and contains base mismatch primer SEQ ID NO.6, SEQ ID NO.7, on first time pcr amplification basis, carry out the nest-type PRC PGC-1 Gene Partial promoter fragment that increases.Polyacrylamide gel electrophoresis is detected the successful PCR product 2 μ L first time of amplification to add in the 18 μ L pure water, therefrom take out 1 μ L behind the mixing, add 2 μ L, 10 * PCR damping fluid, 0.4 μ L 10mMdNTP, 0.5 μ L Taq archaeal dna polymerase, 0.3 μ L sense primer (SEQ ID NO.6,10pmol/ μ L), 0.3 μ L antisense primer (SEQ ID NO.7,10pmol/ μ L), 15.5 μ L pure water, making cumulative volume is 20 μ L.The PCR reaction conditions is 95 ℃ of pre-sex change of 3min, 94 ℃ of 45s, 58 ℃ of 45s, 72 ℃ of 45s, 30 circulations, and 72 ℃ are extended 7min.After reaction was finished, with 3 μ L PCR reaction solutions electrophoresis on 8.0% polyacrylamide gel, Bio-Rad gel imaging system Gel Doc2000 detected, and is the fragment of 193bp as amplification, has obtained single band, is and increases successfully.

5) measure PGC-1 gene promoter region-1998 a SNP polymorphism by handle the PCR reaction product with restriction enzyme.Detect amplification from polyacrylamide gel electrophoresis and draw 3 μ L the PCR product the successful second time, add 1 μ L10 * K restriction enzyme reaction damping fluid, 0.3 μ L restriction enzyme BamH I, pure water 5.7 μ L digest 4h behind the mixing in 37 ℃ of water-baths.Afterwards, on 8.0% polyacrylamide gel, carry out electrophoresis.Bio-Rad gel imaging system Gel Doc2000 detects, and occurs the band of 175bp and 18bp when being C allelotrope as the PGC-1 promotor 1998 point.As when being T allelotrope, band of 193bp only appears, see shown in Figure 2.

Embodiment 3 detection kit

1) preparation detects the test kit of PGC-1 promoter region-1998 (C/G) SNP mark, include four primers that can amplify the PGC-1 Gene Partial promoter sequence that contains-1998 (C/G) SNP mark, and PCR-RFLP corresponding reagent, composition and content following (10 person-portion), in-20 ℃ of preservations:

50 μ L, 10 * PCR damping fluids (Pharmacia),

50 μ L 10mM dNTP mixed solutions (Pharmacia),

10 μ L (2unit/ μ L) Taq archaeal dna polymerase (TAKARA),

Each 5 μ L (10pmol/ μ L) SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 primer,

The 1ml pure water,

20 μ L, 10 * K restriction enzyme reaction damping fluids (TAKARA),

5 μ L (10u/ μ L) restriction enzyme BamH I (TAKARA).

2) according to-1998 (C/G) somatotype conceptual design in the PGC-1 gene promoter sequence among the present invention, use in the present embodiment the detection box to Chinese northerner group totally 396 routine T2DM patients carry out the experiment of mass-producing somatotype.According to test kit experiment somatotype result, 5 examples of sampling in every kind of genotype individuality are individual, get its nest-type PRC the first step PCR and (use primer SEQ ID NO.4, SEQ ID NO.5) after product 20 μ L, behind desalting and purifying, by Beijing China big-and-middle living development in science and technology company limited sequence verification, carry out quality control.The Quality Control result shows; PGC-1 promoter sequence-1998 (C/G) the SNP marker site of all samples; through test kit gene genotyping result consistent with the determining nucleic acid sequence result (seeing shown in Figure 3), show that this test kit confidence level is higher, can be used for promoting and the mass-producing application.

The present invention has the illustration of practicality:

1) found a kind of-1998 (C/G) SNP molecule markers in the Chinese northern crowd PGC-1 gene promoter that are present among the present invention, and design provides the method to this SNP Markers for Detection and special somatotype.The PGC-1 gene is positioned at huamn autosomal 4p15.1, the SNP of this new identification is arranged in the-1998 of PGC-1 gene promoter sequences, find that there are two kinds of multiple allelomorphoss of C/G in this site, can directly apply to or make up the Disease-causing gene location screening that is applied to carry out among the Chinese northern crowd metabolism syndrome behind the haplotype, and may apply to the individual ill risk of metabolism syndrome, be beneficial to carry out the early intervention and the treatment of metabolism syndrome.

2) the PGC-1 gene promoter sequence-1998 bit base variation that utilizes the present invention to set forth, apply to the DNA chip product as molecule marker, merge the SNP molecule marker that other PGC-1 gene regions exist, detect many SNP of PGC-1 gene region mark sudden change situation fast, comprehensive full the effect estimated the metabolism syndrome nosetiology meaning that the PGC-1 gene may exist in specific crowd.

3) the PGC-1 gene promoter sequence-1998 bit base variation that utilizes the present invention to set forth, as one of biomarker, the screening of the molecular target of useful as drug design is regulated the bioactive molecule that PGC-1 expresses to help to seek to have, and promotes the metabolism syndrome new drug development.

4) nucleotide sequence and the specific design of primers of the detection PGC-1 gene promotor 1998 point SNP polymorphism of the present invention's foundation, but highly sensitive is applied to PGC-1 gene promotor 1998 point SNP mark parting kit specifically.

The present invention has narrated the New SNP marker of PGC-1 gene promoter sequence-1998 C/G who exists among the Chinese northern crowd, and the method for the New SNP marker polymorphism of a kind of PGC-1 of mensuration gene promoter sequence-1998 C/G is provided.And, according to the present invention, only need a small amount of DNA sample just to be enough to measure the polymorphism of PGC-1 gene promoter sequence-1998 a C/G SNP mark.As a result, the invention provides the method for the New SNP marker of a kind of PGC-1 of mensuration gene promoter sequence-1998 C/G.

Sequence table

＜110〉Beijing Hospital

＜120〉a kind of primer, method and test kit of identifying the PGC-1 gene promotor 1998 point SNP molecular mark

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<210>6

<211>22

<212>DNA

＜213〉synthetic

<400>6

cagtagtatt?tcacagtgga?tc 22

<210>7

<211>22

<212>DNA

＜213〉synthetic

<400>7

aataacagga?aaatacaacc?tc 22

Claims (4)

1. an isolating nucleic acid is the base sequence shown in the sequence table SEQ ID NO.1.

2. one group of primer of identifying SNP molecule marker in peroxisome proliferation-activated receptors γ co-activator 1 gene (PGC-1 gene) promoter sequence, it is characterized in that: be the base sequence shown in the SEQ ID No.4-SEQ ID No.7, SEQ ID No.4 and SEQ ID No.5 are that primer is right, and SEQ ID No.6 and SEQ ID No.7 are that primer is right.

3. identify SNP molecular marker method in the PGC-1 gene promoter sequence for one kind, it is characterized in that: by extracting the genomic dna of host cell, adopt the method for nest-type PRC, measure experimenter's PGC-1 gene promotor 1998 point SNP marker genotypes in conjunction with restriction site introducing PCR;

Wherein the primer sequence of nest-type PRC is sequence table SEQ ID No.4 and SEQ ID No.5;

The primer sequence that restriction site is introduced PCR is sequence table SEQ ID No.6 and SEQ ID No.7.

4. test kit of identifying SNP molecule marker in the PGC-1 gene promoter sequence, it is characterized in that: be made up of following reagent, storage temperature is-20 ℃:

50 μ L, 10 * PCR damping fluid,

50 μ L 10mM dNTP mixed solutions,

10 μ L Taq archaeal dna polymerases, 2unit/ μ L,

Each 5 μ L SEQ ID NO.4, SEQ IDNO.5, SEQ ID NO.6, SEQ ID NO.7 primer, 10pmol/ μ L,

The 1ml pure water,

20 μ L, 10 * K restriction enzyme reaction damping fluid,

5 μ L restriction enzyme BamH I, 10u/ μ L.

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