CN103060329B - Detection reagent and method for predicting susceptibility to prostate cancer - Google Patents

Abstract

The invention discloses a detection reagent and a method for predicting susceptibility to prostate cancer, belonging to the field of biotechnology. The detection reagent and the method can be used for predicting susceptibility of a subject to prostate cancer by extracting the genomic deoxyribonucleic acid (DNA) of host cells and testing the genotype of the rs2274911 site on the second exon of GPRC 6A gene of the subject; when the genotype of the rs2274911 on the second exon of the GPRC 6A gene is TT, the susceptibility of the subject to prostate cancer is the highest; and when the genotype of the rs2274911 is TC or CC, the susceptibility of the subject to prostate cancer is lower. The detection reagent and the method have the advantages that the correlation between rs2274911 gene polymorphic site and prostate cancer is expounded for the first time; the method for predicting the susceptibility to prostate cancer is provided; and the method can be used for early prevention diagnosis and auxiliary diagnosis of prostate cancer, and is also used for research and development of new medicines.

Description

A kind of detection reagent and method predicting susceptibility to prostate cancer

Technical field

The present invention relates to a kind of detection reagent and the method for predicting susceptibility to prostate cancer, by measuring with relating to prostate cancers because rs2274911 loci polymorphism on the Second Exon of GPRC6A predicts the susceptibility of experimenter for prostate cancer in particular, 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) occurs in modal malignant tumour in male reproductive system.The U.S., after adding up 1975-2006 cancer morbidity, estimates that PCa new cases in 2010 are 217,730 examples, and occupy the male sex and to swell well in knurl first, the death toll of PCa is estimated as 32,050, in male tumor mortality ratio, occupy second.Age is the important risk factor of PCa, and the basic seldom morbidity at less than 45 years old age, increases with the growth at age.American Cancer Society adds up, the male sex PCa sickness rate of less than 40 years old be 1/8499,40-59 year the male sex in be be then 1/8 in more than 1/15, the 70 years old male sex in 1/38,60-69 years old male sex.Along with our countries population's aging, the sickness rate of PCa rises just year by year in China, Shanghai Standardized incidence rate rises to 5.5/10 ten thousand in 1997 ~ 1999 years from 1.8/10 ten thousand in 1973 ~ 1975 years, the sickness rate of part counties and cities, southern areas, Guizhou Province PCa has risen to 4.28/10 ten thousand in 2004 ~ 2008 years from 1.72/10 ten thousand in 1994 ~ 1998 years, become the common problem threatening China's men's health.

Carry out the research of PCa inherited pathogenic factor at present, checking in the extensive genome-wide association study of many employings and the many crowds of small sample, and multiple PCa susceptible SNPs of GWAS qualification have been replicated and have determined in multiple different crowd, demonstrate the association analysis method validity in PCa genetic research of SNPs as genomic marker.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, 70.1% is had for the conversion between homotype base: as G/A or T/C, 29.1% for occurring in the transversion between purine and pyrimidine in this single base change.SNPs contains the 80-90% of known polymorphism, is modal heritable variation.

Because the selective pressure of existence causes the distribution of SNP in term single gene and whole genome to be ununiformity.SNPs is 4 times of coding region in the quantity of gene non-coding region, and sum can reach 300 ten thousand.SNPs with its density high (average every 1kb just have 1), representative strong (SNPs being arranged in gene internal 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 of SNPs crowd for biallelic marker, can simply with " +/-or 1/0 " direct somatotype) and become good genetic marker.

GPRC6A(G protein linked receptor C family 6 groups of A hypotypes) (G protein-coupled receptor, family C, group 6, member A, GPRC6A) gene is that Wellendorph P etc. found to propose in 2004, encoding g-protein coupled receptors C family 6 groups of A members, g protein coupled receptor (G protein-coupled receptors, GPCRs) be that a class has 7 trans-membrane region, it is the large receptor family by about nearly thousand genes encodings on human genome, growth is comprised in many physiological and pathological processes, hematopoiesis, vasculogenesis, inflammation, mental disorder, play an important role in metabolic trouble and virus infection etc.Research shows that many GPCRs and their part participate in tumor development, if endothelin is by breaking up with GPCRs interaction mediating tissue, grow and cell proliferation, 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, Infiltration and metastasis.Many GPCRs being called as endothelial differentiation gene family are acceptors of Ultrapole L or C18-Sphingosine 1-phosphate, regulate the propagation of cell, migration and existence.As many chemokine receptors, neuropeptide receptor, hormone receptor when adenocarcinoma of lung or 3 phase of adenocarcinoma of lung etc.: PAR2(GPR11), Edg2(GPR2) etc. GPCRs up-regulated; When mammary cancer or 3 phase of mammary cancer, PAR2 and CXCR-4 expresses increases; During PCa, CXCR-3, GPR68 etc. are 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, market is GPCRs or relative signal path close to the drug targets of 30%, its research and development be that numerous disease associates with polymorphism with the variation of GPCRs according to part, and therefore associating of research GPCRs and the tumour polymorphism that comprises GPCRs and tumour can for disturb the medicament research and development based on GPCRs or its signal path mediated to provide theoretical foundation.

The existing research of the function of GPRC6A gene, GPRC6A wide expression in brain and 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 kidney process calcium phosphorus is abnormal, fatty liver, and sugar tolerance and steroid synthesize the proterties such as disorderly.Nearest vivo and vitro GPRC6A functional study also shows that it is a target molecule regulating that PCa growth is relevant with tumor development.Moreover GPRC6A is the gene regulating Bone Gla protein to reply in bone-pancreatic secretion ring, bone-pancreatic secretion ring regulates insulin signaling pathway, and insulin receptor and rhIGF-1 are also proved to be relevant to PCa in much research.Hormone can be regulated from GPRC6A and affect insulin signaling pathway these two aspects and support that the variation in site gene may affect the susceptibility of PCa.

2010, Takata R etc. identified rs339331 and the PCa risk association be 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 have selected the rs2274911 being positioned at its exon, in 273 PCa patients and 606 contrast crowds, carry out association analysis after somatotype, result determines rs2274911 and associates with Chinese population PCa.

Rs2274911 on chromosome position is 117,237,396, be positioned on the Second Exon of GPRC6A, this exon has 304 bp, rs2274911 to be the SNP site of holding 76bp apart from this exon 5 ', and this place, site genomic locations may cause transcript mRNA, and translated amino acid sequence changes, and then affect protein functionating.Through query and search, so far, GPRC6A gene rs2274911 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 detecting susceptibility to prostate cancer gene.

Second object of the present invention is to provide a kind of reagent detecting susceptibility to prostate cancer gene, comprises PCR primer and the test kit containing 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 as nucleotide fragments GPRC6A gene Second Exon comprising single nucleotide polymorphism site rs2274911.Fig. 1 is the schematic diagram in GPRC6A gene structure and rs2274911 polymorphic variation site, and GPRC6A gene includes 6 exons, and rs2274911 site is marked on the 6th exon corresponding position in GPRC6A gene map.

When the genotype of described single nucleotide polymorphism site rs2274911 is TT, susceptibility to prostate cancer is the highest; When genotype is TC or CC, susceptibility to prostate cancer is lower.

The primer of one group of detection susceptibility to prostate cancer, can increase and obtain the nucleotide sequence of detection susceptibility to prostate cancer GPRC6A gene Second Exon comprising single nucleotide polymorphism site rs2274911.

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

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

(1) genomic dna of extracting sample, amplification GPRC6A gene Second Exon comprises the nucleotide fragments of single nucleotide polymorphism site rs2274911;

(2) genotype of single nucleotide polymorphism site rs2274911 in detecting step (1) product, when genotype is TT, susceptibility to prostate cancer is the highest; When genotype is TC or 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, and rs2274911 site is positioned at+501 of this nucleotide sequence.

The nucleotide fragments comprising single nucleotide polymorphism site rs2274911 of described amplification GPRC6A gene Second Exon, the nucleotide sequence of one group of primer of use is respectively shown in sequence table SEQ ID No.2 and SEQ ID No.3.

The invention provides a kind of diagnostic kit detecting susceptibility to prostate cancer, the general components, reagent, damping fluid etc. of the primer pair wherein containing specific amplification GPRC6A gene extron rs2274911 site of the present invention and the test kit for pcr amplification detection, those skilled in the art know these general components and detection method.Whole components in test kit of the present invention, content, source and using method are as follows:

Predict a test kit of PCa, detect application for 10 person-portions, be made up of following reagent:

10 μ L 10 × PCR damping fluid (purchased from Pharmacia),

2 μ L 10mM dNTP mixed solution (purchased from Pharmacia),

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

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

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

8 μ L 10 × LC-green PLUS saturated fluorescence dyestuffs; (purchased from American Idaho company)

The each 0.5 μ L of 2 μ L oligonucleotide internal reference primer, concentration is 10pmol/ μ L, wherein low temperature internal reference primers F is the nucleotide sequence shown in SEQ ID NO.4, low temperature internal reference primer R is the nucleotide sequence shown in SEQ ID NO.5, high temperature internal reference primers F is the sequence shown in SEQ ID NO.6, and high temperature internal reference primer R is the nucleotide sequence shown in SEQ ID NO.7;

64 μ L pure water.

Using method:

1) pcr amplification: by the exon Partial Fragment of pcr amplification GPRC6A gene, prepare mixed solution: 10 × PCR reaction buffer 1 μ L, 10mmol/L dNTP 0.2 μ L, Taq archaeal dna polymerase 0.2 μ L, 10pmol/L upstream primer 0.1 μ L, 10pmol/L downstream primer 0.1 μ L, 10 × LC Green PLUS saturated fluorescence dyestuff 0.8 μ L, the each 0.05 μ L of oligonucleotide internal reference 0.2 μ L(high and low temperature oligonucleotide internal reference upstream and downstream primer) (sequence is in table 1), genomic dna 1 μ L, adds deionized water to 10 μ L.PCR reaction conditions is 95 DEG C of denaturation 5min, 95 DEG C of sex change 1min, and 54 DEG C of annealing 30s, 72 DEG C extend 6s, altogether 45 circulations, 72 DEG C of total elongation 7min.Before carrying out high-resolution fusion curve analysis, carry out denature and renature process: 95 DEG C of 30s, 25 DEG C of 2min, 94 DEG C of 30s, 24 DEG C of 4min.In each system, the paraffin oil of 20 μ l is added, to prevent from evaporating during PCR.

2) genotype judges: PCR primer moved in special 96 orifice plates of HRM, Light scanner TMHR-I 96 carries out HRM analysis, analyze the curve after collection with Light Scanner Call IT software, the difference according to melting curve judges genotype.

The purposes of GPRC6A gene Second Exon list nucleotide polymorphism site rs2274911 in the reagent preparing diagnosis or treatment prostate cancer or medicine.

Measuring method of the present invention determines the genomic dna deriving from people, and sample does not limit, as: body fluid (blood, ascites and urine etc.), histocyte (as hepatic tissue) etc.Genomic dna can be prepared by these samples of Isolation and purification.The concentration of adjustment genomic dna, makes it consistent as much as possible.Take genomic dna as template, the nucleic acid fragment containing GPRC6A exons mutation site rs2274911 can be amplified, to obtain the great amount of samples of mensuration.This sample obtained containing the DNA fragmentation of GPRC6A exons mutation point by amplification, is particularly suitable for as mensuration material.

When carrying out gene auxiliary diagnosis, the present invention is preferably applied in the auxiliary diagnostic measuring and exist according to GPRC6A exons mutation site rs2274911 mutation type, auxiliary diagnostic comprises the particular agent as neccessary composition, and it corresponds to the method for measuring rs2274911 gene mutation type.Suitable particular agent is selected, as DNA fragmentation and/or the primer for pcr amplification step by the measuring method adopted.

Advantage of the present invention is: the present invention illustrates the dependency of GPRC6A exon rs2274911 pleomorphism site and PCa first, provide a kind of method and the detection kit of predicting PCa susceptibility, the method can be used for prevention, the auxiliary diagnosis of PCa, can also be used 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; not limitation of the present invention, the equivalent replacement of all any this areas of doing according to the disclosure of invention, all belongs to protection scope of the present invention.

Accompanying drawing explanation

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

Fig. 2 is that GPRC6A gene extron rs2274911 site is through HRM somatotype solubility curve figure

Fig. 3 is the sequencer map in GPRC6A gene extron rs2274911 site

Embodiment

As follows for representing the english abbreviation of reagent in the following example:

10 × PCR damping fluid: 10mM Tris-HCI (pH=8.3), 500mM Repone K (KCI), 10mM magnesium chloride (MgCI2), 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 the PCa patient 273 example (age: 46 – 93 years old from Beijing and Efficiency in Buildings in Tianjin Area consanguinity-less relation altogether, average 72.3 years old), with the contrast 606 example (age: 58 – 94 years old of the age-matched in area, average 70.4 years old), be the male sex, the negative and PSA<4ng/mL without PCa family history, DRE.All persons under inspection are Han nationality and signature Written informed consent, and this research obtains Beijing Hospital, and the accreditation 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 puts upside down mixing 3-5 time before adding), put upside down mixing, standing 10 minutes of room temperature; 2. 13000rpm is after centrifugal 30 seconds, removing supernatant liquor; 3. in gained precipitation, add 480 μ l nucleic acid cleavage liquid, attack tube wall, fully adding 20 μ L Proteinase Ks (diluting 20 times of diluted protein enzyme K with splitting karyolymph) after mixing, putting upside down mixing, 65 DEG C of incubators 10 minutes, (period mixes frequently up and down, guarantees without grumeleuse); 4. be down to room temperature after taking out, add 300 μ L albumen precipitation liquid, fully put upside down mixing, leave standstill 10 minutes, centrifugal 2 minutes of 13000rpm; 5. moved to by supernatant liquor in new EP pipe, add the Virahol of 670 μ L precoolings, fully put upside down mixing (more than 10 times), visible linear DNA forms little agglomerate gradually, centrifugal 2 minutes of 13000rpm; 6. abandon supernatant liquor and guarantee that precipitation is stayed in EP, adding 670 μ L 70% ethanol, mixing of turning upside down, centrifugal 2 minutes of 13000rpm; 7. abandon supernatant, make ethanol volatilization in pipe clean; 8. add TE lysate (400 μ L), fully dissolve, then the genomic dna extracted is carried out to the analysis of concentration and purity, draw part DNA solution as working fluid, concentration correction to 20ng/ μ L, be positioned over 4 DEG C for subsequent use, residue genomic dna put-20 DEG C of Refrigerator stores.

The identification qualification of embodiment 2 SNP

The present invention adopts PCR-high resolving power solubility curve (HRM) analytical method and PCR sequencing technologies to detect the genotype in the rs2274911 site (its loci is T/C) in the exon genes district of GPRC6A simultaneously.

1) determination of PCR-HRM primer

From Genebank, look into the DNA base sequence (SEQ ID NO.1) got near rs2274911, design of primers completes under Oligo 6.0 and primer5.0 software.Object fragment is positioned at GPRC6A gene extron subarea, and total length 72bp determines positive-sense strand F1(+450bp--+467bp) and antisense strand R1(+504bp--+521bp), specific primer sequence is as follows:

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

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

2) PCR-HRM reaction system and condition

By place, the rs2274911 site fragment in the exon genes district of pcr amplification GPRC6A, PCR reaction system is: 10 × PCR reaction buffer 1 μ L, 10mmol/L dNTP 0.2 μ L, Taq archaeal dna polymerase 0.2 μ L, 10pmol/L upstream primer 0.1 μ L, 10pmol/L downstream primer 0.1 μ L, 10 × LC Green PLUS saturated fluorescence dyestuff 0.8 μ L, oligonucleotide internal reference 0.2 μ L(is high, on low temperature oligonucleotide internal reference, the each 0.05 μ L of downstream primer, 3 ' end C3 closes, stop and extend, in table 1), genomic dna 1 μ L, add deionized water to 10 μ L.In each system, add the paraffin oil of 20 μ l during PCR, prevent from evaporating.PCR reaction conditions is 95 DEG C of denaturation 5min, 95 DEG C of sex change 1min, and 54 DEG C of annealing 30s, 72 DEG C extend 6s, altogether 45 circulations, 72 DEG C of total elongation 7min.Before carrying out high-resolution fusion curve analysis, carry out denature and renature process: 95 DEG C of 30s, 25 DEG C of 2min, 94 DEG C of 30s, 24 DEG C of 4min.

Table 1 high and low temperature oligonucleotide internal reference primer sequence, annealing temperature and product sheet segment length

3) HRM judges genotype

PCR primer is moved in special 96 orifice plates of HRM, Light scanner TMHR-I 96 carries out HRM analysis: from 45 DEG C, melting curve is gathered with the slope of 0.3 DEG C/s, to 98 DEG C of end, with Light Scanner Call IT software, the curve (Fig. 2) after collection is analyzed, judge genotype.

4) sequence verification

From the individuality of the different genotype of gained, randomly draw 3 routine samples respectively carry out sequence verification.Order-checking sample re-starts pcr amplification, and sequencing primer sequence is: F2:5 '-GAAATTGCTATGAGGAGATG-3 ' (SEQ ID No.8), R2:5 '-CAGAACCTATGACAGCCTT-3 ' (SEQ ID No.9), the long 339bp of amplified fragments.PCR reaction is totally 30 μ L, comprises: genomic dna 3 μ L, 10 × PCR Buffer 3 μ L, 10mmol/L dNTP 0.6 μ L, Taq archaeal dna polymerase (5U/ μ L) 0.6 μ L, upstream and downstream primer (10pmol/ μ L) each 0.3 μ L, deionized water is supplemented to cumulative volume 30 μ L.PCR reaction conditions is: 95 DEG C of denaturation 5min laggard enter major cycle, 95 DEG C of sex change 30s, 60 DEG C of annealing 30s, 72 DEG C extend 60s, 35 circulations, 72 DEG C of extension 7min.PCR primer detects through 8% polyacrylamide gel electrophoresis, send Hua Da gene sequencing portion to carry out sequence verification (Fig. 3) after gel imaging system observation is qualified.

The dependency of embodiment 3 gene SNP and PCa

Statistical method: the populational representation using Hardy-Weinberg balance check research sample.SPSS11.0 software Pearson chi square test is utilized to calculate allelotrope, the distribution frequency of genotype between PCa case group and Normal group of GPRC6A gene extron rs2274911 pleomorphism site, logistic returns the risk OR value and 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 the SNP rs2274911 polymorphic site of GPRC6A gene extron and gene frequency and refer to table 2 with the association analysis of PCa.

The genotype of table 2GPRC6A gene extron SNP rs2274911 pleomorphism site and the distribution of gene frequency in Chinese population between case-control group and the association analysis with PCa

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

From table 2, the T allelotrope in the rs2274911 site of GPRC6A gene extron, namely be A allelotrope on its DNA complementary strand, distribution frequency in PATIENT POPULATION is significantly higher than its distribution frequency (57.4%vs.51.0%) in healthy normal population, there is significant difference (ageadjustment P=0.014), and the OR value in T site is 1.29,95%CI:1.05-1.59; In recessive model (T/T vs.C/C+T/C), the distribution frequency of its genotype in case group is significantly higher than (ageadjustment P=0.016) in control group, allelotrope and genotype and PCa association analysis all show that the T allelotrope in the rs2274911 site of GPRC6A gene extron 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 rs2274911 site that can amplify GPRC6A gene extron, and other PCR-HRM corresponding reagent.Test kit of the present invention detects application for 10 person-portions, and keep in Dark Place in-20 DEG C, its component, content and source comprise:

10 μ L 10 × PCR damping fluid (Pharmacia),

2 μ L 10mM dNTP mixed solution (Pharmacia),

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

1μL?F1（SEQ?ID?NO.2）（10pmol/μL），

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

8 μ L 10 × LC-green PLUS saturated fluorescence dyestuff (American I daho company),

2 μ L oligonucleotide internal references (10pmol/ μ L) (each 0.5 μ L of high and low temperature oligonucleotide internal reference upstream and downstream primer), (sequence is in table 1),

64 μ L pure water.

Proof test: adopt this test kit, random selecting PCa clinical samples 10 example, control group sample 10 example, detects the polymorphism in GPRC6A gene extron rs2274911 site through PCR-HRM.

1.PCR increases: by the exon Partial Fragment of pcr amplification GPRC6A gene, prepare mixed solution: 10 × PCR reaction buffer 1 μ L, 10mmol/L dNTP 0.2 μ L, Taq archaeal dna polymerase 0.2 μ L, 10pmol/L upstream primer 0.1 μ L, 10pmol/L downstream primer 0.1 μ L, 10 × LC Green PLUS saturated fluorescence dyestuff 0.8 μ L, the each 0.05 μ L of oligonucleotide internal reference 0.2 μ L(high and low temperature oligonucleotide internal reference upstream and downstream primer) (sequence is in table 1), genomic dna 1 μ L, adds deionized water to 10 μ L.PCR reaction conditions is 95 DEG C of denaturation 5min, 95 DEG C of sex change 1min, and 54 DEG C of annealing 30s, 72 DEG C extend 6s, altogether 45 circulations, 72 DEG C of total elongation 7min.Before carrying out high-resolution fusion curve analysis, carry out denature and renature process: 95 DEG C of 30s, 25 DEG C of 2min, 94 DEG C of 30s, 24 DEG C of 4min.In each system, the paraffin oil of 20 μ l is added, to prevent from evaporating during PCR.

2. genotype judges: PCR primer moved in special 96 orifice plates of HRM, Light scanner TMHR-I 96 carries out HRM analysis, analyze the curve after collection with Light Scanner Call IT software, the difference according to melting curve judges genotype.

Two. result:

Result shows, and the genotype in PCa clinical samples GPRC6A gene extron rs2274911 site is TT; The genotype in control group sample rs1321366 site is TC or CC.

The present invention has the illustration of practicality:

The detection method of the rs2274911 polymorphism of GPRC6A gene extron of the present invention can be used for the T loci in this site on analyst's genomic dna, namely be A loci on its DNA complementary strand, be applied to the complementary diagnosis to PCa, individuality can be assessed and have great PCa risk, be beneficial to early prevention and the treatment of carrying out PCa.

The present invention is utilized to set forth the nucleotide variation in the rs2274911 site of GPRC6A gene extron, as one of biomarker, can be used as the screening of the molecular target of medicinal design, to help to find the bioactive molecule having and regulate GPRC6A genetic expression, promote PCa new drug development.

The nucleotide sequence of rs2274911 loci polymorphism of the detection GPRC6A gene extron that the present invention sets up, can highly sensitive, the specific test kit being applied to PCa gene auxiliary diagnosis.

As above told, reached a conclusion, the polymorphism in the rs2274911 site of GPRC6A gene extron and PCa tool significant correlation.Therefore, measure this polymorphism according to the present invention, can be used for the gene auxiliary diagnosis of PCa.

Invention describes the new mutant site that GPRC6A gene extron PCa is relevant, and provide a kind of method measuring gene pleiomorphism, and, according to the present invention, only need a small amount of DNA sample to be just enough to measure the rs2274911 polymorphism of GPRC6A gene extron.

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

Claims (2)

1. one group is detected the primer of susceptibility to prostate cancer, it is characterized in that: the nucleotide sequence of primer is respectively shown in sequence table SEQ ID No.2 and sequence table SEQ ID No.3.

2. detect a test kit for prostate cancer tumor susceptibility gene, it is characterized in that being made up of following reagent:

10 μ L10 × PCR damping fluids;

2 μ L10mM dNTP mixed solutions;

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

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

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

8 μ L10 × LC-green PLUS saturated fluorescence dyestuffs;

The each 0.5 μ L of 2 μ L oligonucleotide internal reference primer, concentration is 10pmol/ μ L, wherein low temperature internal reference primers F is the nucleotide sequence shown in SEQ ID NO.4, low temperature internal reference primer R is the nucleotide sequence shown in SEQ ID NO.5, high temperature internal reference primers F is the sequence shown in SEQ ID NO.6, and high temperature internal reference primer R is the nucleotide sequence shown in SEQ ID NO.7;

64 μ L pure water.