CN103060433B - Kit and method for predicating susceptibility to prostate cancer - Google Patents

Abstract

The invention discloses a kit and a method for predicating the susceptibility to prostate cancer and belongs to the technical field of biology. The susceptibility of a testee to prostate cancer is predicated through extracting a genomic DNA (Deoxyribonucleic Acid) of a host cell and measuring the genotype of an rs1606365 site on a first intron of a GPRC6A gene of the testee; the susceptibility of the testee is the highest when the genotype of the rs1606365 site on the first intron of the GPRC6A gene is CG or GG; and the susceptibility of the testee is the lowest when the genotype of the rs1606365 site is CC. The invention firstly explains the relevancy between a polymorphic site of an rs1606365 gene and the prostate cancer and provides the method for predicating the susceptibility to prostate cancer; and the method can be used for early prevention and diagnosis and auxiliary diagnosis of the prostate cancer and can also be used for new drug research and development.

Description

A kind of test kit and method of predicting susceptibility to prostate cancer

Technical field

The present invention relates to a kind of test kit and method of predicting susceptibility to prostate cancer, say more specifically by measuring with relating to prostate cancers because rs1606365 loci polymorphism prediction experimenter on the intron of GPRC6A is for the susceptibility of prostate cancer, the method can be used for auxiliary diagnosis and the new drug development of disease, belongs to biological technical field.

Background technology

Prostate cancer (prostate cancer, PCa) is to occur in modal malignant tumour in male reproductive system.The U.S., after 1975-2006 cancer morbidity is added up, estimates that PCa new cases in 2010 are 217,730 examples, occupies the male sex and swells well in knurl first, and the death toll of PCa is estimated as 32,050, in male tumor mortality ratio, occupies second.Age is an important risk factor of PCa, and the substantially seldom morbidity that 45 years old age is following increases with the growth at age.American Cancer Society statistics, 40 years old following male sex PCa sickness rate is in 1/8499,40-59 year male sex to be 1/38, is in 1/15,70 years old above male sex to be 1/8 in 60-69 year male sex.Along with our national aging population, the sickness rate of PCa rises just year by year in China, upper seamark sickness rate from 1973～1975 years 1.8/10 ten thousand rise to 1997～1999 years 5.5/10 ten thousand, the sickness rate of the PCa of part counties and cities, southern areas, Guizhou Province from 1994～1998 years 1.72/10 ten thousand rise to 2004～2008 years 4.28/10 ten thousand, become the common problem that threatens China's men's health.

Carry out at present the research of PCa inherited pathogenic factor, the checkings that adopt in extensive genome-wide association study and the many crowds of small sample more, and a plurality of PCa susceptible SNPs that GWAS identifies have been replicated definite in a plurality of different crowds, proved SNPs as the association analysis method of genomic marker the validity in PCa genetic research.SNPs refers to the DNA sequence polymorphism that in chromogene group level, single nucleotide diversity causes, frequency in crowd needs >1%, SNPs is biallelic marker, during changing, this single base has 70.1% for the conversion between homotype base: as G/A or T/C, 29.1% for occurring in the transversion between purine and pyrimidine.The 80-90% that SNPs has comprised known polymorphism is modal heritable variation.

Because the selective pressure of surviving causes the distribution of SNP in term single gene and whole genome to be ununiformity.The quantity of SNPs in gene non-coding region is 4 times of coding region, and sum can reach 300 ten thousand.SNPs take its density high (average every 1kb just has 1), representative strong (SNPs that is arranged in gene inside may directly affect protein structure or expression level), genetic stability good (with microsatellite polymorphism comparatively speaking), be easy to the features such as automated analysis (because SNPs be biallelic marker crowd, can simply with " +/-or 1/0 " direct somatotype) and become good genetic marker.

6 groups of A hypotypes of GPRC6A(G protein linked receptor C family) (G protein-coupled receptor, familyC, group6, memberA, GPRC6A) gene is that Wellendorph P etc. found to propose in 2004, 6 groups of A members of encoding g-protein coupled receptors C family, g protein coupled receptor (G protein-coupled receptors, GPCRs) be that a class has cross-film region 7 times, by the large receptor family of approximately nearly thousand genes encodings on human genome, in many physiological and pathological processes, comprise growth, hematopoiesis, vasculogenesis, inflammation, mental disorder, in metabolic trouble and virus infection etc., play an important role.Research shows that many GPCRs and their part participate in tumor development, if endothelin is by interacting and mediate tissue differentiation, growth and cell proliferation with GPCRs, by strengthening cell inhibitory effect apoptosis and regulating matrix components to play an important role in PCa and other tumours.Many Chemokine Receptors, if CXCR4, CXCR2, CCR7 etc. are at many tumour up-regulateds, play an important role in tumor growth, invasion and attack and transfer.Many GPCRs that are called as endothelial differentiation gene family are acceptors of Ultrapole L or C18-Sphingosine 1-phosphate, regulate propagation, migration and the existence of cell.As adenocarcinoma of lung or adenocarcinoma of lung many chemokine receptors, neuropeptide receptor, hormone receptor etc. during 3 phase: PAR2(GPR11), Edg2(GPR2) etc. GPCRs up-regulated; Mammary cancer or mammary cancer is PAR2 and CXCR-4 expression increase during 3 phase; During PCa, CXCR-3, GPR68 etc., at PCa tissue expression higher than normal prostate tissue, also have multiple GPCRs to participate in the development of other tumour cancer of the stomach, transfer melanoma etc.

According to estimates, on market, approaching 30% drug targets is GPCRs or relative signal path, its research and development be that numerous disease is associated with variation and the polymorphism of GPCRs according to part, so study GPCRs and tumour and comprise that the associated of the polymorphism of GPCRs and tumour can provide theoretical foundation as basic medicament research and development for take the signal path of interference GPCRs or its mediation.

The existing research of function of GPRC6A gene, GPRC6A wide expression Nao He peripheral tissues, high expression level in testis, kidney, skeletal muscle.The mouse that GPRC6A knocks out shows as metabolism syndrome: the bone calcification of scleroblast defect mediation, and it is abnormal that kidney is processed calcium phosphorus, fatty liver, sugar tolerance and steroid synthesize the proterties such as disorderly.Nearest vivo and vitro GPRC6A functional study also shows that it is one and regulates the PCa growth target molecule relevant with tumor development.Moreover GPRC6A is the gene that regulates Bone Gla protein to reply in bone-pancreatic secretion ring, bone-pancreatic secretion ring regulates insulin signaling pathway, and insulin receptor is also proved to be relevant to PCa with rhIGF-1 in much research.From GPRC6A, can regulate hormone and affect this two aspect of insulin signaling pathway supports the variation in site gene may affect the susceptibility of PCa.

2010, Takata R etc. identified rs339331 and the PCa risk association being positioned on RFX6 gene, and after this, checking is copied in this site in European crowd and Chinese population.In conjunction with the above analysis to GPRC6A function, we infer that GPRC6A gene is PCa tumor susceptibility gene and the rs1606365 that has selected to be positioned at its First Intron, in 273 PCa patients and 606 contrast crowds, after somatotype, carry out association analysis, result has determined that rs1606365 is associated with Chinese population PCa.

Rs1606365 is set to 117 karyomit(e) is upper, 238,735, be positioned on the First Intron of GPRC6A, this intron has 19202 bp, rs1606365 is the SNP site apart from initiating terminal 17940, and intron is played an important role at alternative splicing, and therefore a gene can produce multiple different protein.Through query and search, so far, GPRC6A gene rs1606365 and PCa risk yet there are no report.

Summary of the invention

Main purpose of the present invention is to provide a kind of method that detects susceptibility to prostate cancer gene.

Second object of the present invention is to provide a kind of reagent that detects susceptibility to prostate cancer gene, comprises PCR primer and the test kit that contains this primer.

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

Detecting a nucleotide sequence for susceptibility to prostate cancer, is nucleotide sequence shown in sequence table SEQ ID No.1.Described nucleotides sequence is classified the nucleotide fragments that comprises single thuja acid pleomorphism site rs1606365 on GPRC6A gene intron as.Fig. 1 is the schematic diagram in GPRC6A gene structure and rs1606365 polymorphic variation site, and GPRC6A gene includes 5 introns, and rs1606365 site is marked on the corresponding position of the 1st intron in GPRC6A gene map.

When the genotype of described single thuja acid pleomorphism site rs1606365 is CG or GG, susceptibility to prostate cancer is the highest; When genotype is CC, susceptibility to prostate cancer is lower.

One group of primer and probe that detects susceptibility to prostate cancer, the nucleotide sequence of the detection susceptibility to prostate cancer that obtains comprising single thuja acid pleomorphism site rs1606365 on GPRC6A gene intron of increasing.

The nucleotide sequence of described primer and probe is respectively shown in sequence table SEQ ID No.2, sequence table SEQ ID No.3 and sequence table SEQ ID No.4.

A detection method for susceptibility to prostate cancer gene, comprises the steps:

(1) genomic dna of extracting sample, the nucleotide fragments that comprises single thuja acid pleomorphism site rs1606365 on amplification GPRC6A gene intron;

(2) genotype of single thuja acid pleomorphism site rs1606365 in detecting step (1) product, when genotype is CG or GG, susceptibility to prostate cancer is the highest; When genotype is CC, susceptibility to prostate cancer is lower.

Nucleotide fragments in described step (1) is the nucleotide sequence shown in sequence table SEQ ID No.1, rs1606365 site be positioned at this nucleotide sequence+501.

The nucleotide fragments that comprises single thuja acid pleomorphism site rs1606365 of described amplification GPRC6A gene intron, one group of primer of use and the nucleotide sequence of probe are respectively shown in sequence table SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4.

The invention provides a kind of diagnostic kit that detects susceptibility to prostate cancer, the conventional assembly of the test kit that wherein contains the primer pair in specific amplification GPRC6A gene intron rs1606365 of the present invention site and detect for pcr amplification, reagent, damping fluid etc., those skilled in the art know these conventional assembly and detection methods.Whole components, content, source and using method in test kit of the present invention are as follows:

Predict a test kit of PCa, for 10 person-portions, detect application, by following reagent, formed:

10 μ L10 * PCR damping fluids (purchased from Pharmacia),

2 μ L10mM dNTP mixed solutions (purchased from Pharmacia),

2 μ L Taq archaeal dna polymerases (2unit/ μ L) (purchased from Takara),

0.2 μ L F1 primer, is the nucleotide sequence shown in SEQ ID NO.2, and concentration is 10pmol/ μ L;

1 μ L R1 primer, is the nucleotide sequence shown in SEQ ID NO.3, and concentration is 10pmol/ μ L;

1 μ L P probe, is the nucleotide sequence shown in SEQ ID NO.4, and concentration is 10pmol/ μ L;

10 μ L10 * LC-greenPLUS saturated fluorescence dyestuffs; (purchased from American I daho company)

63.8 μ L pure water.

Using method:

1) pcr amplification: by the intron Partial Fragment of pcr amplification GPRC6A gene, prepare mixed solution: 10 * PCR reaction buffer, 1 μ L, 10mmol/L dNTP0.2 μ L, TaqDNA polysaccharase 0.2 μ L, 10pmol/L upstream primer 0.02 μ L, 10pmol/L downstream primer 0.1 μ L, 10 * LC Green PLUS saturated fluorescence dyestuff, 1 μ L, probe 0.1 μ L, genomic dna 1 μ L, adds deionized water to 10 μ L.PCR reaction conditions is 95 ℃ of denaturation 5min, 95 ℃ of sex change 1min, and 54 ℃ of annealing 30s, 72 ℃ are extended 6s, 45 circulations altogether, 72 ℃ of total elongation 7min.Before carrying out high-resolution fusion curve analysis, carry out denature and renature processing: 95 ℃ of 30s, 25 ℃ of 2min, 94 ℃ of 30s, 24 ℃ of 4min.During PCR, in each system, add the paraffin oil of 20 μ l, evaporate preventing.

2) genotype is judged: PCR product is moved in special-purpose 96 orifice plates of HRM, on Light scanner TMHR-I96, carry out HRM analysis, with Light Scanner Call IT software, the curve after gathering is analyzed, according to the difference of melting curve, judged genotype.

Use probe cold but that 3 ' end has C3 to seal, LCgreenplusDNA combination dye can be combined in the two strands of probe and pairing object chain formation, a DNA chain (chain of being combined with probe) is wherein that excessive amplification makes to produce too much object chain, if SNPs is homozygous, object chain only has a kind of form, but wild isozygoty with sudden change isozygoty different from the tightness degree of probe combination, while unwinding, respectively form one unimodal; If SNPs is heterozygosis, the object chain of amplification has two kinds of forms, and two kinds of forms of probe combination form bimodal while determining to unwind, thereby three kinds of different genotype are distinguished.

GPRC6A gene intron list thuja acid pleomorphism site rs1606365 is in preparation diagnosis or the reagent for the treatment of prostate cancer or the purposes in medicine.

Measuring method of the present invention has been measured the genomic dna that derives from people, and sample is restriction not, as: body fluid (blood, ascites and urine etc.), histocyte (as hepatic tissue) etc.By extraction and these samples of purifying, can prepare genomic dna.The concentration of adjusting genomic dna, makes it consistent as much as possible.Take genomic dna as template, can amplify the nucleic acid fragment containing GPRC6A intragenic mutation site rs1606365, to obtain the great amount of samples of mensuration.This sample that contains the DNA fragmentation acquisition of GPRC6A intragenic mutation point by amplification, is particularly suitable for as measuring material.

When carrying out gene auxiliary diagnosis, the present invention is preferably applied in and measures the auxiliary diagnostic existing according to GPRC6A intron rs1606365 mutation type, auxiliary diagnostic comprises the particular agent as neccessary composition, and it is corresponding to for measuring the method for rs1606365 gene mutation type.By the measuring method adopting, select suitable particular agent, as DNA fragmentation and/or for the primer of pcr amplification step.

Advantage of the present invention is: the present invention has illustrated the dependency of GPRC6A intron rs1606365 pleomorphism site and PCa first, a kind of method and test kit of the PCa of prediction susceptibility are provided, the method can be used for prevention, the auxiliary diagnosis of PCa, can also be for new drug development.

Below in conjunction with the drawings and specific embodiments, the present invention is further described; so that the public has a better understanding summary of the invention; limitation of the present invention not, all any this areas of doing according to the disclosure of invention be equal to replacement, all belong to protection scope of the present invention.

Accompanying drawing explanation

Fig. 1 is the schematic diagram in GPRC6A gene structure and intron polymorphic variation site

Fig. 2 is that GPRC6A gene intron rs1606365 site is through HRM somatotype solubility curve figure

Fig. 3 is the sequencer map in GPRC6A gene intron rs1606365 site

Embodiment

For the following example, represent that the english abbreviation of reagent is as follows:

10 * PCR damping fluid: 10mM Tris-HCl (pH=8.3), 500mM Repone K (KCl), 10mM magnesium chloride (MgCl ₂), 0.01% (W/V) gelatinum

DNTP: deoxynucleoside triphosphate

EDTA: disodium ethylene diamine tetraacetate

TE：10mM?Tris-HCI(pH=7.5)，1mM?EDTA(pH=8.0)

Embodiment 1: the extraction of blood sample collection and genomic dna:

(1) PCa patient is all through histopathologic diagnosis, choose altogether the PCa patient's 273 examples (age: 46 – 93 years old from Beijing and Efficiency in Buildings in Tianjin Area consanguinity-less relation, average 72.3 years old), with the contrast 606 examples (age: 58 – 94 years old of regional age-matched, average 70.4 years old), be the male sex, without PCa family history, DRE feminine gender and PSA<4ng/mL.All persons under inspection are Han nationality and sign written Informed Consent Form, and this research obtains Beijing Hospital, and the approval of ethics audit committee of Gerontological Research Center institute of the Ministry of Health, meets AMM's Declaration of Helsinki: the ethic principle of human medical research.(2) according to following method, preparation human gene group DNA.1. first in the 1.5mLEP pipe of label, add 1000 μ L erythrocyte cracked liquids, after add 400 μ LEDTA anticoagulations (anticoagulation is put upside down and mixed 3-5 time before adding), put upside down and mix, standing 10 minutes of room temperature; 2. 13000rpm, after centrifugal 30 seconds, removes supernatant liquor; 3. in gained precipitation, add 480 μ l nucleic acid lysates, attack tube wall, adds 20 μ L Proteinase Ks (diluting 20 times of diluted protein enzyme K with splitting karyolymph) after fully mixing, and puts upside down and mixes, 65 ℃ of incubators 10 minutes, (during frequently mix up and down, guarantee without grumeleuse); 4. after taking out, be down to room temperature, add 300 μ L albumen precipitation liquid, fully put upside down and mix, standing 10 minutes, centrifugal 2 minutes of 13000rpm; 5. supernatant liquor is moved in new EP pipe, add the Virahol of 670 μ L precoolings, fully put upside down and mix (more than 10 times), visible linear DNA forms little agglomerate, centrifugal 2 minutes of 13000rpm gradually; 6. abandon supernatant liquor and guarantee to precipitate and stay in EP, adding 670 μ L70% ethanol, turning upside down and mix, centrifugal 2 minutes of 13000rpm; 7. abandon supernatant, make to manage interior ethanol volatilization clean; 8. add TE lysate (400 μ L), fully dissolve, then the genomic dna extracting is carried out to the analysis of concentration and purity, draw part DNA solution as working fluid, concentration correction is to 20ng/ μ L, be positioned over 4 ℃ standby, residue genomic dna is put-20 ℃ of Refrigerator stores.

The identification of embodiment 2SNP is identified

The present invention adopts PCR-high resolving power solubility curve (HRM) analytical method and PCR sequencing technologies the genotype of the rs1606365 site of the intron gene regions of GPRC6A (its loci is C/G) to be detected simultaneously.

1) PCR-HRM primer determines

From Genebank, look near the DNA base sequence (SEQ ID NO.1) of getting rs1606365, design of primers completes under Oligo6.0 and primer5.0 software.Object fragment is positioned at GPRC6A gene intron district, and total length 180bp has determined positive-sense strand F1(+425bp--+446bp) and antisense strand R1(+583bp--+604bp), specific primer sequence is as follows:

F1：5’-AGAAGTAAGCCCAGCTCATCAT-3’（SEQ?ID?NO.2）

R1：5’-AGGTGCCTTACTCAAATACTGA-3’（SEQ?ID?NO.3）

P：5’-CCTGTCACTGCCGTTGCCATC-3’（SEQ?ID?NO.4）

2) PCR-HRM reaction system and condition

Place, the rs1606365 site fragment of the intron gene regions by pcr amplification GPRC6A, PCR reaction system is: 10 * PCR reaction buffer, 1 μ L, 10mmol/L dNTP0.2 μ L, Taq archaeal dna polymerase 0.2 μ L, 10pmol/L upstream primer 0.02 μ L, 10pmol/L downstream primer 0.1 μ L, 10 * LC Green PLUS saturated fluorescence dyestuff, 1 μ L, probe 0.1 μ L, genomic dna 1 μ L, adds deionized water to 10 μ L.During PCR, in each system, add the paraffin oil of 20 μ l, prevent from evaporating.PCR reaction conditions is 95 ℃ of denaturation 5min, 95 ℃ of sex change 1min, and 54 ℃ of annealing 30s, 72 ℃ are extended 6s, 45 circulations altogether, 72 ℃ of total elongation 7min.Before carrying out high-resolution fusion curve analysis, carry out denature and renature processing: 95 ℃ of 30s, 25 ℃ of 2min, 94 ℃ of 30s, 24 ℃ of 4min.

3) HRM judges genotype

PCR product is moved in special-purpose 96 orifice plates of HRM, on Light scanner TMHR-I96, carry out HRM analysis: since 45 ℃, slope with 0.3 ℃/s gathers melting curve, to 98 ℃ of end, with Light ScannerCall IT software, the curve (Fig. 2) after gathering is analyzed, judged genotype.

4) sequence verification

From the individuality of the different genotype of gained, randomly draw respectively 3 routine samples and carry out sequence verification.Order-checking sample re-starts pcr amplification, and sequencing primer sequence is: F2:5 '-AGAAGTAAGCCCAGCTCATCAT-3 ' (SEQ ID No.5), R2:5 '-AGGTGCCTTACTCAAATACTGA-3 ' (SEQ ID No.6), the long 180bp of amplified fragments.PCR reaction is totally 30 μ L, comprises: genomic dna 3 μ L, 10 * PCRBuffer3 μ L, 10mmol/L dNTP0.6 μ L, Taq archaeal dna polymerase (5U/ μ L) 0.6 μ L, each 0.3 μ L of upstream and downstream primer (10pmol/ μ L), deionized water is supplemented to cumulative volume 30 μ L.PCR reaction conditions is: 95 ℃ of denaturation 5min are laggard enters major cycle, 95 ℃ of sex change 30s, and 60 ℃ of annealing 30s, 72 ℃ are extended 60s, 35 circulations, 72 ℃ are extended 7min.PCR product detects through 8% polyacrylamide gel electrophoresis, after gel imaging system observation is qualified, send Hua Da gene sequencing portion to carry out sequence verification (Fig. 3).

The dependency of embodiment 3 gene SNPs and PCa

Statistical method: use the colony of Hardy-Weinberg balance check research sample representative.Utilize SPSS11.0 software Pearson chi square test to calculate allelotrope, the distribution frequency of genotype between PCa case group and Normal group of GPRC6A gene intron rs1606365 pleomorphism site, logistic returns ill risk OR value and the 95%CI credibility interval thereof of evaluating PCa, take P<0.05 as significance of difference standard.

Result: be positioned at the distribution between case and control group of the genotype of SNP rs1606365 polymorphic site of GPRC6A gene intron and gene frequency and refer to table 2 with the association analysis of PCa.

The genotype of table 1GPRC6A gene intron SNP rs1606365 pleomorphism site and gene frequency are in China

Distribution in crowd between case-control group and with the association analysis of PCa

Note: OR: odds ratio; CI: credibility interval.HWE:Hardy-Weinberg balance.

From table 1, the G allelotrope in the rs1606365 site of GPRC6A gene intron, on its DNA complementary strand, be C allelotrope, distribution frequency in patient colony is significantly higher than its distribution frequency (46.2%vs.38.4%) in healthy normal population, there is significant difference (ageadjustment P=0.002), and the OR value in G site is 1.39,95%CI:1.13-1.71, in recessive model (G/Gvs.C/C+C/G), the distribution frequency of its genotype in case group is significantly higher than (the ageadjustment P=0.002) in control group, in dominant models (G/G+C/Gvs.C/C), the distribution frequency of its genotype in case group is also significantly higher than (the P=0.017 in control group, OR:1.46, 95%CI:1.07-1.98, ageadjustment P=0.035), allelotrope and genotype and PCa association analysis all show that the G allelotrope in the rs1606365 site of GPRC6A gene intron may be proportionate with PCa is ill, likely increase the onset risk of PCa.

Embodiment 4 detection kit

Preparation detects the test kit of PCa relevant risk, includes the primer pair in the rs1606365 site that can amplify GPRC6A gene intron, and other PCR-HRM corresponding reagent.Test kit of the present invention detects application for 10 person-portions, keeps in Dark Place in-20 ℃, and its component, content and source comprise:

10 μ L10 * PCR damping fluids (Pharmacia),

2 μ L10mM dNTP mixed solutions (Pharmacia),

2 μ L TaqDNA polysaccharases (2unit/ μ L) (Takara),

0.2μL?F1（SEQ?ID?NO.2）（10pmol/μL），

1 μ L R1(SEQ ID NO.3) (10pmol/ μ L) primer, (the raw work in Shanghai is synthetic)

1 μ L P probe, is the nucleotide sequence shown in SEQ ID NO.4, and concentration is 10pmol/ μ L; (the raw work in Shanghai is synthetic)

10 μ L10 * LC-green PLUS saturated fluorescence dyestuffs (American I daho company),

63.8 μ L pure water.

Proof test: adopt this test kit, choose at random PCa clinical samples 10 examples, control group sample 10 examples, the polymorphism through PCR-HRM detection GPRC6A gene intron rs1606365 site.

One. method

1.PCR amplification: by the intron Partial Fragment of pcr amplification GPRC6A gene, prepare mixed solution: 10 * PCR reaction buffer, 1 μ L, 10mmol/L dNTP0.2 μ L, TaqDNA polysaccharase 0.2 μ L, 10pmol/L upstream primer 0.02 μ L, 10pmol/L downstream primer 0.1 μ L, probe 0.1 μ L, 10 * LC Green PLUS saturated fluorescence dyestuff, 1 μ L, genomic dna 1 μ L, adds deionized water to 10 μ L.PCR reaction conditions is 95 ℃ of denaturation 5min, 95 ℃ of sex change 1min, and 54 ℃ of annealing 30s, 72 ℃ are extended 6s, 45 circulations altogether, 72 ℃ of total elongation 7min.Before carrying out high-resolution fusion curve analysis, carry out denature and renature processing: 95 ℃ of 30s, 25 ℃ of 2min, 94 ℃ of 30s, 24 ℃ of 4min.During PCR, in each system, add the paraffin oil of 20 μ l, evaporate preventing.

2. genotype is judged: PCR product is moved in special-purpose 96 orifice plates of HRM, on Light scanner TMHR-I96, carry out HRM analysis, with Light Scanner Call IT software, the curve after gathering is analyzed, according to the difference of melting curve, judged genotype.

Use probe cold but that 3 ' end has C3 to seal, LCgreenplusDNA combination dye can be combined in the two strands of probe and pairing object chain formation, a DNA chain (chain of being combined with probe) is wherein that excessive amplification makes to produce too much object chain, if SNPs is homozygous, object chain only has a kind of form, but wild isozygoty with sudden change isozygoty different from the tightness degree of probe combination, while unwinding, respectively form one unimodal; If SNPs is heterozygosis, the object chain of amplification has two kinds of forms, and two kinds of forms of probe combination form bimodal while determining to unwind, thereby three kinds of different genotype are distinguished.

Two. result:

Result demonstration, the genotype in PCa clinical samples GPRC6A gene intron rs1606365 site is CG or GG; The genotype in control group sample rs1321366 site is CC.

The present invention has the illustration of practicality:

The detection method of the rs1606365 polymorphism of GPRC6A gene intron of the present invention can be used for the G loci in this site on analyst's genomic dna, on its DNA complementary strand, be C loci, be applied to the complementary diagnosis to PCa, can assess individuality has the ill risk of great PCa, is beneficial to carry out early prevention and the treatment of PCa.

Utilize the present invention to set forth the nucleotide variation in the rs1606365 site of GPRC6A gene intron, as one of biomarker, the screening of the molecular target of useful as drug design, has to help to find the bioactive molecule that regulates GPRC6A genetic expression, promotes PCa new drug development.

The nucleotide sequence of the rs1606365 loci polymorphism of the detection GPRC6A gene intron that the present invention sets up, can highly sensitive, the specific test kit that is applied to PCa gene auxiliary diagnosis.

As above tell, reach a conclusion, the polymorphism in the rs1606365 site of GPRC6A gene intron and PCa tool significant correlation.Therefore, according to the present invention, measure this polymorphism, can be used for the gene auxiliary diagnosis of PCa.

The present invention has narrated the relevant new mutant site of GPRC6A gene intron PCa, and a kind of method of measuring gene pleiomorphism is provided, and, according to the present invention, only need a small amount of DNA sample to be just enough to measure the rs1606365 polymorphism of GPRC6A gene intron.

The invention provides a kind of gene aided diagnosis method of the PCa of mensuration related gene polymorphism.

Claims (1)

1. detect a test kit for prostate cancer tumor susceptibility gene, it is characterized in that being formed by following reagent:

10 μ L10 * PCR damping fluids;

2 μ L10mM dNTP mixed solutions;

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

0.2 μ L F1 primer, is the nucleotide sequence shown in SEQ ID No.2, and concentration is 10pmol/ μ L;

1 μ L R1 primer, is the nucleotide sequence shown in SEQ ID No.3, and concentration is 10pmol/ μ L;

1 μ L P probe, is the nucleotide sequence shown in SEQ ID NO.4, and concentration is 10pmol/ μ L;

10 μ L10 * LC-green PLUS saturated fluorescence dyestuffs;

63.8 μ L pure water.