CN103060315B - Detection kit and method for predicting susceptibility to prostate cancer - Google Patents

Abstract

The invention discloses a detection kit and a method for predicting susceptibility to prostate cancer, belonging to the field of biotechnology. The detection kit 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 rs4714476 site near 5' end of FOXP 4 gene of the subject; when the genotype of the rs4714476 near 5' end of the FOXP 4 gene is CC or TC, the susceptibility of the subject to prostate cancer is higher; when the genotype of the rs4714476 is TT, the susceptibility of the subject to prostate cancer is the lowest; and the clinical phenotype of the prostate cancer of the subject can be predicted. The occurrence rates of neoplasm staging III and advanced prostate cancer of the patients with the genotype of CC or TC are apparently higher than those of the patients with the genotype of TT. The detection kit and the method have the advantages that the correlation between rs4714476 gene polymorphic site and prostate cancer as well as the clinical information of 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 kit and method predicting susceptibility to prostate cancer

Technical field

The present invention relates to a kind of detection kit and the method for predicting susceptibility to prostate cancer, by to measure and relating to prostate cancers predicts the susceptibility of experimenter for prostate cancer because of rs4714476 loci polymorphism near FOXP4 gene 5 ' end 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) is the modal malignant tumour relevant to old-age group of the male sex, and its pathogenesis it be unclear that, and family history and twin study show that inherited genetic factors is the important risk factor that PCa occurs.Along with the growth of China aging populations, the sickness rate of PCa increases (Liu Zhenwei just year by year, Xiang Yongbing, Zhang Wei. 1973 ~ 1999 years PCa analysis of pathogenetic tendencies in Areas in Shanghai City. China Health is added up, 2003, 20 (6): 335-337.), current PCa specific antigens (prostate specific antigen, PSA) application in PCa examination causes numerous dispute, existing PCa examination is the detection relying on PSA level again more, find clinically, due to specificity and the susceptibility deficiency of PSA accuracy of detection, bring the underdiagnosis of PCa and the serious problems of over-treatment.So have not yet to see the research of the clinical early screening PCa technology of substituting PS A, reason lacks effective PCa biological marker.Research shows that the PCa early diagnosis that is found to be of increasing PCa genetic marker provides possibility.

Along with the appearance of extensive genome-wide association study (Genome-Wide Association Studies, GWAS), identify more than 50 PCa susceptible SNPs site by this method abroad at present, about explain the PCa inherited pathogenic factor of 25%.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.C(cytosine(Cyt)) be the most labile site in human genome because great majority are methylated cytosines, can spontaneous deaminizating be converted to T(thymus pyrimidine), 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.

FOXP4(jaw transcription factor P4) (forkhead box P4, FOXP4) gene is the P subfamily of FOX transcription factor family, play a major role in fetal development, Cycle Regulation and tumour are formed, in wild mouse tissue and mankind tumor tissue, FOXP4 expresses and obviously reduces in patients with renal cell carcinoma tissue, and the research such as Frohme et al. also proves that the expression of the FOXP4 when laryngocarcinoma also reduces.Demonstrate the dependency of FOXP4 gene and tumour.At present, the effect possible in PCa of FOXP4 is also not studied, but in the mammary cancer similar to PCa pathogenesis and genetic mechanism, the variation or the dystopy that have identified FOXP4 gene change.

PCa susceptible SNP site rs1983891 on FOXP4 gene is the GWAS research qualifications in Japanese population such as Takata R in 2010, FOXP4 gene is positioned at 6p21, rs1983891 is positioned at FOXP4 gene intron 2, the cognation of this site and PCa is in Europe at present, checking is replicated in Chinese population, and the risk of FOXP4 gene and other sites neighbouring and PCa yet there are no and studies have reported that, we are in conjunction with the above FOXP4 function relevant with other tumours, in this gene and gene two ends 5KB, select SNPs to carry out somatotype and analyze the dependency with PCa and PCa relevant clinical phenotype, identify and be positioned at the relevant clinical information association of rs4714476 and PCa near FOXP4 gene 5 ' end and PCa.

Rs4714476 to be positioned on 6p near FOXP4 gene 5 ' end, FOXP4 full length gene 55.96kbp, on 6p21, position is from 41,622,142 to 41,678,099, rs4714476 on chromosome position is 41,621,717, distance FOXP4 gene only 425bp, may have regulating effect to FOXP4 genetic transcription, or the chain functional site having transcription initiation region, through query and search, so far, the risk that there is not yet rs4714476 loci polymorphism and PCa near FOXP4 gene 5 ' end has 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 the nucleotide fragments comprising single nucleotide polymorphism site rs4714476 near FOXP4 gene 5 ' end.Fig. 1 is the schematic diagram in FOXP4 gene structure and rs4714476 polymorphic variation site, and rs47144768 site is marked on the corresponding position in FOXP4 gene map near 5 ' end.

When the genotype of described single nucleotide polymorphism site rs4714476 is CC or TC, susceptibility to prostate cancer is the highest; When genotype is TT, susceptibility to prostate cancer is lower.This genotype Site discrepancy is measurable experimenter's prostate cancer clinical phenotypes also: compared with normal control population, when near FOXP4 gene 5 ' end, the genotype of rs4714476 is CC or TC, diagnosis of age >=65 year old in patients with prostate cancer, PSA(PCa specific antigens) horizontal >20ng/ml, neoplasm staging <III and the incidence of advancing prostate carcinoma all will apparently higher than the genotypic patient of TT.

The primer of one group of detection susceptibility to prostate cancer, can increase and obtain the nucleotide sequence of detection susceptibility to prostate cancer according to claim 1.

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, comprises the nucleotide fragments of single nucleotide polymorphism site rs4714476 near amplification FOXP4 gene 5 ' end;

(2) genotype of single nucleotide polymorphism site rs4714476 in detecting step (1) product, when genotype is CC or TC, susceptibility to prostate cancer is the highest; When genotype is TT, 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 rs4714476 site is positioned at+301 of this nucleotide sequence.

Comprise the nucleotide fragments of single nucleotide polymorphism site rs4714476 near described amplification FOXP4 gene 5 ' end, 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 test kit detecting prostate cancer tumor susceptibility gene, the general components, reagent, damping fluid etc. of the primer pair wherein containing rs4714476 site near specific amplification FOXP4 gene 5 ' end 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;

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 SEQID 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 Partial Fragment near pcr amplification FOXP4 gene 5 ' end, 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 single nucleotide polymorphism site rs4714476 in the reagent preparing diagnosis or treatment prostate cancer or medicine near FOXP4 gene 5 ' end.

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 mutational site rs4714476 near FOXP4 gene 5 ' end can be amplified, to obtain the great amount of samples of mensuration.This by the sample of amplification containing the DNA fragmentation acquisition in mutational site near FOXP4 gene 5 ' end, be particularly suitable for being used as to measure material.

When carrying out gene auxiliary diagnosis, the present invention is preferably applied in the auxiliary diagnostic measuring and exist according to mutational site rs4714476 mutation type near FOXP4 gene 5 ' end, auxiliary diagnostic comprises the particular agent as neccessary composition, and it corresponds to the method for measuring rs4714476 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 rs4714476 pleomorphism site and PCa near FOXP4 gene 5 ' end first, provide a kind of method and the detection reagent of predicting PCa susceptibility, the method can be used for auxiliary diagnosis and the new drug development of PCa.

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 end polymorphic variation site near FOXP4 gene structure and 5 '

Fig. 2 holds rs600928 site through HRM somatotype solubility curve figure near FOXP4 gene 5 '

Fig. 3 is the sequencer map in end rs600928 site near FOXP4 gene 5 '

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 (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 patient is all through histopathologic diagnosis, choose the PCa patient 277 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 602 example (age: 58 – 94 years old of the age-matched in area, average 70.5 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 300mL 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 2SNP

The present invention adopts PCR-high resolving power solubility curve (HRM) analytical method and PCR sequencing technologies to detect the genotype in the rs4714476 site (its loci is C/T) of gene regions near FOXP4 gene 3 ' end simultaneously.

1) determination of PCR-HRM primer

From Genebank, look into the DNA base sequence (SEQ ID NO.1) got near rs4714476, design of primers completes under Oligo 6.0 and primer5.0 software.Object fragment is positioned at FOXP4 gene 5 ' near gene district, and total length 61bp, determines positive-sense strand F1(+271bp--+291bp) and antisense strand R1(+315bp--+331bp), specific primer sequence is as follows:

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

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

2) PCR-HRM reaction system and condition

By place, the rs4714476 site fragment of gene regions near the 5 ' end of pcr amplification FOXP4, 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 ScannerCall 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 '-TGGTTCGCAGAGCACTTTACGG-3 ' (SEQ ID No.8), R2:5 '-CCCTGGCCTCGGACTAATCCTA-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 × PCRBuffer3 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, 54 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 rs4714476 pleomorphism site near FOXP4 gene 3 ' end, 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 rs4714476 polymorphic site near FOXP4 gene 5 ' end and gene frequency and refer to table 2 with the association analysis of Pca.

The genotype of SNP rs4714476 pleomorphism site and the distribution of gene frequency between case-control group and the association analysis with PCa near table 2FOXP4 gene 5 ' end

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

From table 2, the C allelotrope in the rs4714476 site near FOXP4 gene 5 ' gene, namely be G allelotrope on its DNA complementary strand, distribution frequency in PATIENT POPULATION is significantly higher than its distribution frequency (37.5%vs.31.6%) in healthy normal population, there is significant difference (ageadjustment P=0.012), and the OR value in C site is 1.32,95%CI:1.06-1.63; In dominant models (C/C+T/C vs.T/T), the distribution frequency of its genotype in case group is significantly higher than (ageadjustment P=0.012) in control group, allelotrope and genotype and PCa association analysis all show that the C allelotrope in the rs4714476 site near FOXP4 gene 5 ' end may be proportionate with PCa is ill, likely increase the onset risk of PCa.

The dependency of embodiment 4 gene SNP and PCa clinical phenotypes

SPSS11.0 software Pearson chi square test is utilized to calculate the distribution frequency of genotype in PCa case group under different clinical phenotypes of rs4714476 pleomorphism site near FOXP4 gene 3 ' end, and compare with normal control population, being returned the risk OR value and 95%CI credibility interval thereof of evaluating the different clinical phenotypes of the PCa patients carrying different genotype by logistic, take P<0.05 as significance of difference standard.

Result: the distribution of the genotype being positioned at the SNP rs4714476 polymorphic site near FOXP4 gene 5 ' end between case and control group and refer to table 3 with the association analysis of PCa clinical phenotypes.

SNP rs4714476 polymorphic site genotype near table 3FOXP4 gene 5 ' end and the association analysis of PCa clinical phenotypes

Note: OR: odds ratio; CI: credibility interval.

From table 3, the CC+TC genotype in the rs4714476 site near FOXP4 gene 5 ' gene, distribution frequency in the different clinical phenotypes of patient higher than its distribution frequency in healthy normal population (diagnosis by exclusion age <65 year, all the other OR>1), compared with normal control population, when near FOXP4 gene 5 ' end, the genotype of rs4714476 is CC or TC, diagnosis of age >=65 year old in patients with prostate cancer, the horizontal >20ng/ml of PSA, neoplasm staging <III and the incidence of advancing prostate carcinoma all will apparently higher than the genotypic patient (p=0.031-0.005 of TT, OR, 1.581-1.989).

Implement 5 detection kit

Preparation detects the test kit of PCa relevant risk, includes the primer pair in the rs4714476 site that can amplify near FOXP4 gene 5 ' end, 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 Tap 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 the rs4714476 site near FOXP4 gene 5 ' end through PCR-HRM.

1.PCR amplification: by Partial Fragment near pcr amplification FOXP4 gene 5 ' end, 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 near PCa clinical samples FOXP4 gene 5 ' end, the genotype in rs4714476 site is CC or TC; The genotype in control group sample rs1321366 site is TT.

When near FOXP4 gene 5 ' end, the genotype of rs4714476 is CC or TC, patients with clinical manifestations is: age >=65 years old, the horizontal >20ng/ml of PSA, neoplasm staging <III, the incidence of advancing prostate carcinoma is high.

The present invention has the illustration of practicality:

The detection method of the rs4714476 polymorphism near FOXP4 gene 5 ' end of the present invention can be used for the C loci in this site on analyst's genomic dna, namely be G loci on its DNA complementary strand, be applied to the complementary diagnosis to PCa, can assess individuality and have great PCa risk, and assessment individuality there is the risk of much performances different PCa relevant clinical phenotype to be beneficial to carry out early prevention and the treatment of PCa.

The present invention is utilized to set forth the nucleotide variation in the rs4714476 site near FOXP4 gene 5 ' end, 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 FOXP4 genetic expression, promote PCa new drug development.

The nucleotide sequence of rs4714476 loci polymorphism near the detection FOXP4 gene 5 ' end 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 rs4714476 site near FOXP4 gene 5 ' end 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 near FOXP4 gene 5 ' end, 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 rs4714476 polymorphism near FOXP4 gene 5 ' end.

The invention provides a kind of gene aided diagnosis method utilizing the loci polymorphism near FOXP4 gene 5 ' to predict susceptibility to prostate cancer.

Claims (1)

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

10 μ L 10 × PCR damping fluids;

2 μ L 10mM 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 L 10 × LC-green PLUS saturated fluorescence dyestuffs;

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

64 μ L pure water.