CN103205487A - Method and detection reagent for prediction of prostate cancer susceptibility - Google Patents

Abstract

The invention discloses a method and a detection reagent for prediction of prostate cancer susceptibility, belonging to the field of biotechnology. According to the invention, genomic DNA of a host cell is extracted and the genotype of site rs600928 located near a terminal 3' of the GPRC6A gene of a subject is determined so as to predicate susceptibility of the subject to a prostate cancer; if the genotype of the rs600928 near the terminal 3' of the GPRC6A gene is CC, the subject has highest susceptibility, and if the genotype of the rs600928 is TC or TT, the subject has low susceptibility. The invention has the following advantages: correlation between the rs600928 gene polymorphism site and the prostate cancer is elaborated for the first time; the method for predicting prostate cancer susceptibility is provided; and the method is applicable to early prevention and diagnosis and auxiliary diagnosis of the prostate cancer and can also be used for research and development of a novel drug.

Description

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

Technical field

The present invention relates to a kind of method and detection reagent of predicting the prostate cancer susceptibility, say so more specifically and predict that because of near the rs600928 loci polymorphism 3 ' end of GPRC6A the experimenter is for the susceptibility of prostate cancer by measuring with relating to prostate cancers, this method can be used for auxiliary diagnosis and the new drug development of disease, belongs to biological technical field.

Background technology

(prostate cancer is to occur in modal malignant tumour in the male reproductive system PCa) to prostate cancer.The U.S. estimates that PCa new cases in 2010 are 217,730 examples after the 1975-2006 cancer morbidity is added up, occupy the male sex and swell in the knurl first well, and the death toll of PCa is estimated as 32,050, occupies second in the male tumor mortality ratio.Age is the important risk factor of PCa, and the substantially seldom morbidity of age below 45 years old increases with the growth at age.American Cancer Society statistics, the male sex PCa sickness rate below 40 years old are to be 1/38 among 1/8499,40-59 year male sex, are then to be 1/8 among the male sex more than 1/15,70 years old among 60-69 year male sex.Along with our national aging population, the sickness rate of PCa rises just year by year in China, last seamark sickness rate from 1973～1975 years 1.8/10 ten thousand rise to the 5.5/10 ten thousand (Liu Zhenwei in 1997～1999 years, Xiang Yongbing, open common vetch. 1973～1999 years PCa incidence trends in urban district, Shanghai are analyzed. Chinese health statistics, 2003,20 (6): 335-337.), 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 (Huang Guijun that threatens China's men's health, Lan Zejun, Quan Xin. the PCa incidence trend research in 1994～2008 years of part counties and cities, southern areas, Guizhou Province. modern preventive medicine, 2011,37 (116): 3012-3017.).

Carry out the research of PCa inherited pathogenic factor at present, genome association study (the Genome-WideAssociation Studies entirely that adopt on a large scale more, GWAS) and the checking among the many crowds of small sample, proved association analysis method the validity in PCa genetic research of SNPs as genomic marker.SNPs refers to the dna sequence polymorphism that single nucleotide diversity causes on the chromogene group level, frequency in the crowd needs＞1%, SNPs is biallelic marker, have 70.1% to be the conversion between the homotype base during this single base changes: as G/A or T/C, 29.1% for occurring in the transversion between purine and the pyrimidine.C (cytosine(Cyt)) is the most labile site in the human genome, because great majority are the cytosine(Cyt)s that methylate, can be converted to T (thymus pyrimidine) by spontaneous deaminizating, and SNPs has comprised 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 height (average every 1kb just has 1), representative strong (SNPs that is arranged in gene inside may directly influence protein structure or expression level), genetic stability good (with microsatellite polymorphism comparatively speaking), be easy to characteristics such as automated analysis (because SNPs be biallelic marker the crowd, can be simply with "+/-or 1/0 " direct somatotype) and become good genetic marker.

GPRC6A (6 groups of A hypotypes of g protein coupled receptor C family) (G protein-coupled receptor, familyC, group 6, member A, GPRC6A) gene is that Wellendorph P etc. found to propose in 2004, coding g protein coupled receptor C 6 groups of A members of family, g protein coupled receptor (G protein-coupled receptors, GPCRs) be that a class has and strides diaphragm area 7 times, be on the human genome by the big receptor family of nearly thousand genes encodings approximately, comprise growth in many physiological and pathological processes, hematopoiesis, vasculogenesis, inflammation, mental disorder, play an important role in metabolic trouble and the virus infection etc.Studies show that many GPCRs and their part participate in tumor development, mediate tissue differentiation, growth and cell proliferation as endothelin by interacting with GPCRs, play an important role in PCa and other tumours by strengthening the cell inhibitory effect apoptosis and regulating matrix components.Many Chemokine Receptors such as CXCR4, CXCR2, CCR7 etc. play an important role in tumor growth, invasion and attack and transfer at many tumour up-regulateds.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.: PAR2 (GPR11), Edg2 GPCRs up-regulateds such as (GPR2) during 3 phases; Mammary cancer or mammary cancer is PAR2 and CXCR-4 expression increase during 3 phases; CXCR-3, GPR68 etc. are higher than normal prostate tissue at the PCa tissue expression during PCa, also have multiple GPCRs to participate in the development of other tumour cancer of the stomach, transfer melanoma etc.

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

The existing research of the function of GPRC6A gene, GPRC6A wide expression is 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 defective mediation, it is unusual that kidney is handled calcium phosphorus, fatty liver, proterties such as sugar tolerance and the synthetic disorder of steroid.Nearest vivo and vitro GPRC6A functional study shows that also it is one and regulates the PCa growth target molecule relevant with tumor development.Moreover GPRC6A regulates the gene that Bone Gla protein is replied in bone-pancreatic secretion ring, and bone-pancreatic secretion ring is regulated insulin signaling pathway, and insulin receptor also is proved to be relevant with PCa with rhIGF-1 in many researchs.The variation that can regulate hormone and influence site on this two aspect of insulin signaling pathway support gene from GPRC6A may influence the susceptibility of PCa.

2010, Takata R etc. identified that the rs339331 that is positioned on the RFX6 gene is related with the PCa risk, and after this, checking is copied in this site in European crowd and Chinese population.Rs339331 is positioned at RFX6 gene the 4th intron, it is positioned at the related section of an about 200kb to cover 580kb zone (Chr.6:117.0-117.6Mb) mapping analysis with 59 tag SNPs, this zone is contained two gene: GPRC6A, RFX6, in conjunction with above analysis to the GPRC6A function, we infer that the GPRC6A gene is the PCa tumor susceptibility gene and has selected to be positioned near the rs600928 of its 3 ' end, carry out association analysis behind the somatotype in 273 PCa patients and 606 contrast crowds, the result has determined that rs600928 is related with Chinese population PCa.

Rs600928 is positioned at 3 ' the near gene end of GPRC6A, karyomit(e) is upper to be set to 117,219,422, apart from last mRNA translation end on this gene 517 bp are arranged, rs600928 is the terminal nearest SNP site of distance 3 ' gene translation, and may there be regulating effect this genome position, place, site to genetic transcription.Through query and search, so far, GPRC6A gene rs600928 and PCa risk yet there are no report.

Summary of the invention

Main purpose of the present invention provides a kind of method that detects the prostate cancer susceptible gene.

Second purpose of the present invention provides a kind of reagent that detects the prostate cancer susceptible 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:

A kind of nucleotide sequence that detects the prostate cancer susceptibility is nucleotide sequence shown in the sequence table SEQ ID No.1.Described nucleotides sequence is classified near the nucleotide fragments that comprises single thuja acid pleomorphism site rs600928 the GPRC6A gene 3 ' end as.The rs600928 site namely is the+366 bit base Y (Y=C or T).Fig. 1 is the synoptic diagram in GPRC6A gene structure and rs600928 polymorphic variation site, and the GPRC6A gene includes 6 exons, and the rs600928 site is marked near the corresponding position the 6th exon in the GPRC6A gene map.

The present invention discovers that when the genotype of described single thuja acid pleomorphism site rs600928 was CC, the prostate cancer susceptibility was the highest; When genotype was TC or TT, the prostate cancer susceptibility was lower.

One group of primer that detects the prostate cancer susceptibility can obtain near the nucleotide fragments that comprises single thuja acid pleomorphism site rs600928 of GPRC6A gene 3 ' end by specific amplification.The nucleotide sequence of described primer is respectively shown in sequence table SEQ ID No.2 and the sequence table SEQ ID No.3.

A kind of detection method of prostate cancer susceptible gene comprises the steps:

(1) genomic dna of extracting sample, near the nucleotide fragments that comprises single thuja acid pleomorphism site rs600928 the amplification GPRC6A gene 3 ' end;

(2) genotype of single thuja acid pleomorphism site rs600928 in detection step (1) product, when genotype was CC, the prostate cancer susceptibility was the highest; When genotype was TC or TT, the prostate cancer susceptibility was lower.

Nucleotide fragments in the described step (1) is the nucleotide sequence shown in the sequence table SEQ ID No.1, the rs600928 site be positioned at this nucleotide sequence+366.

Near the described amplification GPRC6A gene 3 ' end the nucleotide fragments that comprises single thuja acid pleomorphism site rs600928, the nucleotide sequence of one group of primer of use is respectively shown in sequence table SEQ ID No.2 and the SEQ ID No.3.

The invention provides a kind of diagnostic kit that detects the prostate cancer susceptibility, the primer that wherein contains near the rs600928 site specific amplification GPRC6A gene 3 ' gene of the present invention to the conventional assembly that is used for the test kit that pcr amplification detects, reagent, damping fluid etc., those skilled in the art know these conventional assembly and detection methods.Whole components, content, source and using method in the test kit of the present invention are as follows:

A kind of test kit of predicting PCa detects application for 10 person-portions, is made up of following reagent:

10 μ L, 10 * PCR damping fluids (available from Pharmacia),

2 μ L 10mM dNTP mixed solutions (available from Pharmacia),

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

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

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

8 μ L, 10 * LC-green PLUS saturated fluorescence dyestuff; (available from American I daho company)

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

64 μ L pure water.

Using method:

1) pcr amplification: by near the part fragment 3 ' gene of pcr amplification GPRC6A gene, preparation 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 GreenPLUS saturated fluorescence dyestuff, 0.8 μ L, oligonucleotide confidential reference items 0.2 μ L (each 0.05 μ L of high and low temperature oligonucleotide confidential reference items upstream and downstream primer) (sequence sees Table 1), genomic dna 1 μ L adds deionized water to 10 μ L.The PCR reaction conditions is 95 ℃ of pre-sex change 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 the analysis of high resolution melting curve, carry out sex change and renaturation and handle: 95 ℃ of 30s, 25 ℃ of 2min, 94 ℃ of 30s, 24 ℃ of 4min.The paraffin oil that adds 20 μ l during PCR in each system evaporates preventing.

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

Nucleotide sequence shown in the sequence table SEQ ID No.1 and comprise near the GPRC6A gene 3 ' end the nucleotide fragments that comprises single thuja acid pleomorphism site rs600928 in preparation diagnosis or the reagent for the treatment of prostate cancer or the purposes in the medicine.

Near the GPRC6A gene 3 ' end single thuja acid pleomorphism site rs600928 is in preparation diagnosis or the reagent for the treatment of prostate cancer or the purposes in the medicine.

Measuring method of the present invention has been measured the genomic dna that derives from the people, sample without limits, as body fluid (blood, ascites and urine etc.), histocyte (as hepatic tissue) etc.Can prepare genomic dna by extraction and these samples of purifying.Adjust the concentration of genomic dna, make it consistent as much as possible.Be template with the genomic dna, can amplify the nucleic acid fragment that contains near the mutational site rs600928 GPRC6A 3 ' gene, to obtain the great amount of samples of mensuration.Thisly contain the sample that near the dna fragmentation of the catastrophe point GPRC6A 3 ' gene obtains by amplification, be particularly suitable for as measuring material.

When carrying out the gene auxiliary diagnosis, the present invention is preferably applied in the auxiliary diagnostic that mensuration exists according near the mutational site rs600928 mutation type GPRC6A 3 ' gene, auxiliary diagnostic comprises the particular agent as neccessary composition, and it is corresponding to the method that is used for measuring the rs600928 gene mutation type.Select suitable particular agent by the measuring method that adopts, as dna fragmentation and/or be used for the primer of pcr amplification step.

Advantage of the present invention is: the present invention has illustrated the dependency of near the rs600928 pleomorphism site of GPRC6A 3 ' gene and PCa first, a kind of method and test kit of the PCa of prediction susceptibility are provided, this 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 done further narration; so that the public has more deep understanding to summary of the invention; be not limitation of the present invention, 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.

Description of drawings

Fig. 1 is near the synoptic diagram in end polymorphic variation site GPRC6A gene structure and 3 '

Fig. 2 holds the rs600928 site through HRM somatotype solubility curve figure near the GPRC6A gene 3 ' gene

Fig. 3 is near the sequencer map in the end rs600928 site GPRC6A gene 3 ' gene

Embodiment

The english abbreviation that is used for the following example expression 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-HCl(pH＝7.5)，1mM?EDTA(pH＝8.0)

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

1.PCa the patient is all through histopathologic diagnosis, choose the PCa patient's 273 examples (age: 46-93 year from Beijing and Efficiency in Buildings in Tianjin Area consanguinity-less relation altogether, average 72.3 years old), with the contrast 606 examples (age: 58-94 year of the age-matched in area, average 70.4 years old), be the male sex, no 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, prepare the human gene group DNA.1. at first add 1000 μ L erythrocyte cracked liquids in the 1.5mLEP pipe of label, the back adds 400 μ L EDTA anticoagulations (anticoagulation is put upside down mixing 3-5 time before adding), puts upside down mixing, and room temperature left standstill 10 minutes; 2. 13000rpm removes supernatant liquor after centrifugal 30 seconds; 3. add 480 μ l nucleic acid lysates in gained precipitation, the attack tube wall fully adds 20 μ L Proteinase Ks (with splitting 20 times of diluted protein enzyme K of karyolymph dilution) behind the mixing, put upside down mixing, 65 ℃ of incubators 10 minutes, (during frequently about mixing, guaranteeing does not have grumeleuse); 4. be down to room temperature after taking out, add 300mL albumen precipitation liquid, fully put upside down mixing, left standstill 10 minutes, centrifugal 2 minutes of 13000rpm; 5. supernatant liquor is moved in the 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, centrifugal 2 minutes of 13000rpm gradually; 6. abandon supernatant liquor and guarantee the precipitation stay among the EP, add 670 μ L, 70% ethanol, the mixing that turns upside down, centrifugal 2 minutes of 13000rpm; 7. abandon supernatant, make the interior ethanol volatilization of pipe clean; 8. add TE lysate (400 μ L), fully dissolving is carried out the analysis of concentration and purity to the genomic dna that extracts then, draw the part dna solution as working fluid, concentration correction is to 20ng/ μ L, be positioned over 4 ℃ standby, the residue genomic dna is put-20 ℃ of refrigerators and is preserved.

The identification of embodiment 2:SNP is identified

The present invention adopts PCR-high resolving power solubility curve (HRM) analytical method and PCR sequencing technologies simultaneously near the genotype in the rs600928 site (its loci is C/T) of the gene regions 3 ' end of GPRC6A to be detected.

1.PCR-HRM determining of primer

Look near the DNA base sequence of getting the rs600928 (SEQ ID NO.1) from Genebank, design of primers is finished under Oligo 6.0 and primer5.0 software.The purpose fragment is positioned at GPRC6A gene 3 ' near gene district, total length 80bp, determined positive-sense strand F1 (+312bp--+331bp) with antisense strand R1 (+370bp--+391bp), specific primer sequence is as follows:

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

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

2.PCR-HRM reaction system and condition

Place, rs600928 site fragment by near the gene regions 3 ' end of pcr amplification GPRC6A, the PCR reaction system is: 10 * PCR reaction buffer, 1 μ L, 10mmol/L dNTP 0.2 μ L, TaqDNA polysaccharase 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 confidential reference items 0.2 μ L (height, on the low temperature oligonucleotide confidential reference items, each 0.05 μ L of downstream primer), 3 ' end C3 sealing stops and extends, and sees Table 1), genomic dna 1 μ L adds deionized water to 10 μ L.In each system, add the paraffin oil of 20 μ l during PCR, prevent from evaporating.The PCR reaction conditions is 95 ℃ of pre-sex change 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 the analysis of high resolution melting curve, carry out sex change and renaturation and handle: 95 ℃ of 30s, 25 ℃ of 2min, 94 ℃ of 30s, 24 ℃ of 4min.

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

3.HRM judgement genotype

The PCR product is moved in special-purpose 96 orifice plates of HRM, carrying out HRM at Light scanner TMHR-I 96 analyzes: since 45 ℃, slope with 0.3 ℃/s is gathered melting curve, to 98 ℃ of end, with LightScanner Call 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 3 routine samples respectively and carry out sequence verification.The order-checking sample carries out pcr amplification again, and the sequencing primer sequence is: F2:5 '-TGAATGCACCTTCTTGTCG-3 ' (SEQID No.8), R2:5 '-AGGAGAAACAGGCACATATCA-3 ' (SEQ ID No.9), the long 278bp of amplified fragments.The PCR reaction totally is 30 μ L, comprises: genomic dna 3 μ L, 10 * PCR Buffer3 μ L, 10mmol/L dNTP 0.6 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.The PCR reaction conditions is: 95 ℃ of pre-sex change 5min are laggard to go into major cycle, 95 ℃ of sex change 30s, and 60 ℃ of annealing 30s, 72 ℃ are extended 60s, 35 circulations, 72 ℃ are extended 7min.The PCR product detects through 8% polyacrylamide gel electrophoresis, send the big gene sequencing portion of China to carry out sequence verification (Fig. 3) after the gel imaging system observation is qualified.

Embodiment 3: the dependency of gene SNP and PCa

Statistical method: colony's representativeness of using Hardy-Weinberg balance check research sample.Utilize SPSS11.0 software Pearson chi square test to calculate allelotrope, the distribution frequency of genotype between PCa case group and normal control group of near the rs600928 pleomorphism site of GPRC6A gene 3 ' end, logistic returns ill risk OR value and the 95%CI credibility interval thereof of estimating PCa, is significance of difference standard with P＜0.05.

Result: be positioned near the genotype of the SNP rs600928 polymorphic site the GPRC6A gene 3 ' end and the distribution of gene frequency between case and control group and see table 2 for details with the association analysis of Pca.

Near the table 2GPRC6A gene 3 ' end genotype of SNP rs600928 pleomorphism site and gene frequency in

Distribution among the state crowd between the case-control group reaches the association analysis with PCa

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

By table 2 as seen, near the C allelotrope in the rs600928 site the GPRC6A gene 3 ' gene, be G allelotrope at its DNA complementary strand namely, distribution frequency in patient colony is significantly higher than its distribution frequency (58.2%vs.51.2%) in healthy normal population, has significant difference (ageadjustment P=0.006), and the OR value in C site is 1.33,95%CI:1.08-1.64; In recessive model (C/C vs.T/T+T/C), the distribution frequency of its genotype in the case group is significantly higher than (the ageadjustment P=0.001) in the control group, allelotrope and genotype and PCa association analysis show that all near the C allelotrope in the rs600928 site the GPRC6A gene 3 ' end may be proportionate with PCa is ill, might increase the onset risk of PCa.

Embodiment 4: detection kit

Preparation detects the test kit of PCa relevant risk, and it is right to include the primer that can amplify near the rs600928 site of GPRC6A gene 3 ' end, and other PCR-HRM corresponding reagent.Test kit of the present invention detects for 10 person-portions and uses, and keeps in Dark Place in-20 ℃, and its component, content and source comprise:

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

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

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

1 μ L F1 primer (SEQ ID NO.2) (10pmol/ μ L),

1 μ L R1 primer (SEQ ID NO.3) (10pmol/ μ L), (it is synthetic that the worker is given birth in Shanghai)

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

2 μ L oligonucleotide confidential reference items (10pmol/ μ L) (each 0.5 μ L of high and low temperature oligonucleotide confidential reference items upstream and downstream primer), (sequence sees Table 1),

64 μ L pure water.

Proof test: adopt this test kit, picked at random PCa patient sample 10 examples, control group sample 10 examples, near the polymorphism in the rs600928 site PCR-HRM detects GPRC6A gene 3 ' end.

One. method:

1.PCR amplification: by near the part fragment 3 ' gene of pcr amplification GPRC6A gene, preparation 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, oligonucleotide confidential reference items 0.2 μ L (each 0.05 μ L of high and low temperature oligonucleotide confidential reference items upstream and downstream primer) (sequence sees Table 1), genomic dna 1 μ L adds deionized water to 10 μ L.The PCR reaction conditions is 95 ℃ of pre-sex change 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 the analysis of high resolution melting curve, carry out sex change and renaturation and handle: 95 ℃ of 30s, 25 ℃ of 2min, 94 ℃ of 30s, 24 ℃ of 4min.The paraffin oil that adds 20 μ l during PCR in each system evaporates preventing.

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

Two. the result:

The result shows that near the genotype in the rs600928 site PCa patient's sample GPRC6A gene 3 ' end is CC; The genotype in control group sample rs600928 site is TC or TT.

The present invention has the illustration of practicality:

Near the detection method of the rs600928 polymorphism the GPRC6A gene 3 ' end of the present invention can be used for the C loci in this site on analyst's genomic dna, be the G loci at its DNA complementary strand namely, 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 near the nucleotide variation in the rs600928 site of GPRC6A gene 3 ' end, as one of biomarker, the screening of the molecular target of useful as drug design has the bioactive molecule of regulating GPRC6A genetic expression to help to seek, and promotes the PCa new drug development.

Near the nucleotide sequence of the rs600928 loci polymorphism the detection GPRC6A gene 3 ' end that the present invention sets up, but highly sensitive, the specific test kit that is applied to PCa gene auxiliary diagnosis.

As above tell, reach a conclusion, polymorphism and the PCa tool significant correlation near the rs600928 site the GPRC6A gene 3 ' end.Therefore, measure this polymorphism according to the present invention, can be used for the gene auxiliary diagnosis of PCa.

The present invention has narrated near the relevant new mutant site of the PCa of GPRC6A gene 3 ' end, and provide a kind of method of measuring gene pleiomorphism, and, according to the present invention, only need a small amount of DNA sample just to be enough to measure near the rs600928 polymorphism of GPRC6A gene 3 ' end.

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

Claims (10)

1. a nucleotide sequence that detects the prostate cancer susceptibility is characterized in that: be nucleotide sequence shown in the sequence table SEQ ID No.1.

2. the nucleotide sequence of detection prostate cancer susceptibility according to claim 1 is characterized in that: described nucleotides sequence is classified near the nucleotide fragments that comprises single thuja acid pleomorphism site rs600928 the GPRC6A gene 3 ' end as.

3. the nucleotide sequence of detection prostate cancer susceptibility according to claim 2, it is characterized in that: when the genotype of described single thuja acid pleomorphism site rs600928 was CC, the prostate cancer susceptibility was the highest; When genotype was TC or TT, the prostate cancer susceptibility was lower.

4. one group of primer that detects the prostate cancer susceptibility is characterized in that: can increase obtains GPRC6A gene 3 ' and holds near the nucleotide fragments that comprises single thuja acid pleomorphism site rs600928.

5. one group of primer that detects the prostate cancer susceptibility according to claim 4, it is characterized in that: the nucleotide sequence of described primer is respectively shown in sequence table SEQ ID No.2 and the sequence table SEQ ID No.3.

6. the detection method of a prostate cancer susceptible gene is characterized in that: comprise the steps:

(1) genomic dna of extracting sample, near the nucleotide fragments that comprises single thuja acid pleomorphism site rs600928 the amplification GPRC6A gene 3 ' end;

(2) genotype of single thuja acid pleomorphism site rs600928 in detection step (1) product, when genotype was CC, the prostate cancer susceptibility was the highest; When genotype was TC or TT, the prostate cancer susceptibility was lower.

7. the detection method of prostate cancer susceptible gene according to claim 6, it is characterized in that: near the nucleotide fragments that comprises single thuja acid pleomorphism site rs600928 the described amplification GPRC6A gene 3 ' end, the nucleotide sequence of one group of primer of use is respectively shown in sequence table SEQ ID No.2 and the SEQ ID No.3.

8. test kit that detects the prostate cancer tumor susceptibility gene is characterized in that being made up of following reagent:

10 μ L, 10 * PCR damping fluid;

2 μ L 10mM dNTP mixed solutions;

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

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

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

8 μ L, 10 * LC-green PLUS saturated fluorescence dyestuff;

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

64 μ L pure water.

9. nucleotide sequence shown in the sequence table SEQ ID No.1 and comprise near the GPRC6A gene 3 ' end the nucleotide fragments that comprises single thuja acid pleomorphism site rs600928 in preparation diagnosis or the reagent for the treatment of prostate cancer or the purposes in the medicine.

10.GPRC6A near the single thuja acid pleomorphism site rs600928 the gene 3 ' end is in preparation diagnosis or the reagent for the treatment of prostate cancer or the purposes in the medicine.

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