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

Abstract

The invention discloses a kit and a method for predicting susceptibility to prostate cancer, belonging to the field of biotechnology. The present invention measures the genotype of the rs1606365 site on the first intron of the GPRC6A gene of the subject by extracting the genomic DNA of the host cell, and predicts the susceptibility of the subject to prostate cancer; When the genotype was CG or GG, the susceptibility of the subject was the highest; when the genotype of rs1606365 was CC, the susceptibility of the subject was lower. The advantages of the present invention are: the correlation between the rs1606365 gene polymorphism site and prostate cancer is described for the first time, and a method for predicting the susceptibility of prostate cancer is provided, which can be used for early preventive diagnosis and auxiliary diagnosis of prostate cancer, and also It can be used in the development of new drugs.

Description

A kit and method for predicting susceptibility to prostate cancer

technical field

The present invention relates to a kit and method for predicting the susceptibility of prostate cancer, more specifically, predicting the susceptibility of subjects to prostate cancer by determining the rs1606365 site polymorphism on the intron of the prostate cancer-related gene GPRC6A , the method can be used for auxiliary diagnosis of diseases and development of new medicines, and belongs to the field of biotechnology.

Background technique

Prostate cancer (PCa) is the most common malignant tumor occurring in the male reproductive system. After statistics on the incidence of cancer from 1975 to 2006 in the United States, it is estimated that there were 217,730 new cases of PCa in 2010, ranking first among male tumors, and the death toll of PCa is estimated to be 32,050, which is the median of male tumor mortality. Ranked second. Age is an important risk factor for PCa, and it is rare for those under the age of 45 to develop PCa, which increases with age. According to statistics from the American Cancer Society, the incidence rate of PCa is 1/8499 in men under 40 years old, 1/38 in men aged 40-59, 1/15 in men aged 60-69, and 1/8 in men over 70 years old . With the aging population in our country, the incidence rate of PCa is increasing year by year in our country. The standardized incidence rate in Shanghai rose from 1.8/100,000 in 1973-1975 to 5.5/100,000 in 1997-1999. The incidence of PCa in counties and cities has risen from 1.72/100,000 in 1994 to 1998 to 4.28/100,000 in 2004 to 2008, becoming a public problem that threatens the health of men in my country.

At present, the research on the genetic etiology of PCa mostly adopts large-scale genome-wide association studies and verification in small samples and multiple populations, and multiple PCa-susceptible SNPs identified by GWAS have been replicated and confirmed in multiple different populations, which proves that SNPs are the basis of genome Validity of marker association analysis methods in PCa genetic studies. SNPs refer to DNA sequence polymorphisms caused by single nucleotide variation at the chromosomal genome level, and the frequency in the population needs to be >1%. SNPs are biallelic markers, and 70.1% of these single base changes are homotypes Conversion between bases: such as G/A or T/C, 29.1% are transversions between purine and pyrimidine. SNPs contain 80-90% of known polymorphisms and are the most common genetic variation.

The distribution of SNPs in single genes and across the genome is heterogeneous due to selection pressure for survival. The number of SNPs in the non-coding region of genes is 4 times that of the coding region, and the total number can reach 3 million. SNPs are characterized by their high density (1 per 1 kb on average), strong representation (SNPs located inside genes may directly affect protein structure or expression level), good genetic stability (compared to microsatellite polymorphisms), Ease of automatic analysis (because SNPs are biallelic markers in the population, they can be directly typed by "+/- or 1/0") and other characteristics have become good genetic markers.

GPRC6A (G protein-coupled receptor C family 6 group A subtype) (G protein-coupled receptor, familyC, group6, memberA, GPRC6A) gene was discovered and proposed by Wellendorph P in 2004, encoding G protein-coupled receptors C family 6 group A members, G protein-coupled receptors (GPCRs) is a class of 7 transmembrane regions, is a large receptor family encoded by nearly a thousand genes on the human genome, It plays an important role in many physiological and pathological processes including development, hematopoiesis, angiogenesis, inflammation, mental disorders, metabolic diseases and viral infections. Studies have shown that many GPCRs and their ligands are involved in the development of tumors. For example, endothelins mediate tissue differentiation, development and cell proliferation by interacting with GPCRs, inhibit apoptosis and regulate matrix components in PCa and other tumors by enhancing cell proliferation. important role in tumors. Many chemokine receptors, such as CXCR4, CXCR2, CCR7, etc. are up-regulated in many tumors and play an important role in tumor growth, invasion and metastasis. Many GPCRs known as the endothelial differentiation gene family are receptors for lysophosphatidic acid or sphingosine-1-phosphate, regulating cell proliferation, migration and survival. For example, in lung adenocarcinoma or lung adenocarcinoma stage 3, many chemical factor receptors, neuropeptide receptors, hormone receptors, etc.: the expression of GPCRs such as PAR2 (GPR11), Edg2 (GPR2) is up-regulated; breast cancer or breast cancer stage 3 PAR2 The expressions of CXCR-3 and GPR68 in PCa tissue were higher than those in normal prostate tissue in PCa, and a variety of GPCRs were also involved in the development of other tumors such as gastric cancer and metastatic melanoma.

It is estimated that nearly 30% of the drug targets on the market are GPCRs or their related signaling pathways. The research and development is partly based on the association between many diseases and the variation and polymorphism of GPCRs. Therefore, the study of GPCRs and tumors, including the polymorphisms of GPCRs and tumor The association can provide a theoretical basis for the development of drugs based on interference with GPCRs or their mediated signaling pathways.

The function of GPRC6A gene has been studied. GPRC6A is widely expressed in brain and peripheral tissues, and highly expressed in testis, kidney and skeletal muscle. GPRC6A-knockout mice exhibit metabolic syndrome: bone calcification mediated by osteoblast defects, abnormal renal processing of calcium and phosphorus, fatty liver, glucose tolerance, and disturbances in steroidogenesis, among other traits. Recent in vivo and in vitro functional studies of GPRC6A also indicated that it is a target molecule involved in regulating PCa growth and tumor development. Furthermore, GPRC6A is a gene that regulates osteocalcin response in the bone-pancreas secretory loop, which regulates the insulin signaling pathway, and insulin receptor and insulin-like growth factor have also been shown to be associated with PCa in many studies. From the aspects that GPRC6A can regulate hormones and affect the insulin signaling pathway, it is supported that the variation of the site on the gene may affect the susceptibility to PCa.

In 2010, Takata R et al. identified the risk association between rs339331 and PCa on the RFX6 gene, and since then, this site has been replicated and verified in European and Chinese populations. Combined with the above analysis of the function of GPRC6A, we speculated that the GPRC6A gene is a PCa susceptibility gene and selected rs1606365 located in its first intron, and performed association analysis after typing in 273 PCa patients and 606 control populations, and the results confirmed that rs1606365 was associated with PCa in Chinese population.

The position of rs1606365 on the chromosome is 117,238,735, located on the first intron of GPRC6A, the intron has 19202 bp, rs1606365 is the SNP site 17940 away from the starting end, the intron plays an important role in alternative splicing, a Genes can thus produce many different proteins. After searching, up to now, there has been no report on the relationship between GPRC6A gene rs1606365 and the risk of PCa.

Contents of the invention

The main purpose of the present invention is to provide a method for detecting prostate cancer susceptibility genes.

The second object of the present invention is to provide a reagent for detecting prostate cancer susceptibility genes, including PCR primers and a kit containing the primers.

To achieve the above object, the present invention adopts the following technical solutions:

A nucleotide sequence for detecting susceptibility to prostate cancer is the nucleotide sequence shown in SEQ ID No.1 in the sequence table. The nucleotide sequence is a nucleotide fragment including the single nucleotide polymorphism site rs1606365 on the intron of the GPRC6A gene. Figure 1 is a schematic diagram of the structure of the GPRC6A gene and the polymorphic variation site of rs1606365. The GPRC6A gene contains 5 introns, and the rs1606365 site is marked at the corresponding position of the first intron in the GPRC6A gene map.

When the genotype of the single nucleotide polymorphism site rs1606365 is CG or GG, the susceptibility to prostate cancer is the highest; when the genotype is CC, the susceptibility to prostate cancer is low.

A set of primers and probes for detecting prostate cancer susceptibility can amplify and obtain a nucleotide sequence for detecting prostate cancer susceptibility including single nucleotide polymorphism site rs1606365 on the intron of GPRC6A gene.

The nucleotide sequences of the primers and probes are respectively shown in the sequence listing SEQ ID No.2, the sequence listing SEQ ID No.3 and the sequence listing SEQ ID No.4.

A method for detecting prostate cancer susceptibility genes, comprising the steps of:

(1) Extract the genomic DNA of the sample, and amplify the nucleotide fragment containing the single nucleotide polymorphism site rs1606365 on the intron of the GPRC6A gene;

(2) Detection of the genotype of the single nucleotide polymorphism site rs1606365 in the product of step (1). When the genotype is CG or GG, the susceptibility to prostate cancer is the highest; when the genotype is CC, the susceptibility to prostate cancer is low.

The nucleotide fragment in the step (1) is the nucleotide sequence shown in the sequence table SEQ ID No.1, and the rs1606365 site is located at +501 of the nucleotide sequence.

The amplified GPRC6A gene intron contains a nucleotide fragment of the single nucleotide polymorphism site rs1606365, and the nucleotide sequences of a set of primers and probes used are the sequence table SEQ ID No.2, SEQ ID No.2 and SEQ ID No.2 respectively. Shown in ID No.3 and SEQ ID No.4.

The invention provides a diagnostic kit for detecting the susceptibility of prostate cancer, which contains the primer pair for specifically amplifying the GPRC6A gene intron rs1606365 site of the invention and the conventional components and reagents of the kit for PCR amplification detection , buffer, etc. Those skilled in the art are familiar with these conventional components and detection methods. All components, contents, sources and methods of use in the kit of the present invention are as follows:

A test kit for predicting PCa, for 10 human test applications, consists of the following reagents:

10 μL 10×PCR buffer (purchased from Pharmacia),

2 μL 10mM dNTP mixture (purchased from Pharmacia),

2 μL Taq DNA polymerase (2unit/μL) (purchased from Takara),

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

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

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

10 μL 10×LC-greenPLUS saturated fluorescent dye; (purchased from Idaho, USA)

63.8 μL pure water.

Instructions:

1) PCR amplification: Amplify the intron fragment of the GPRC6A gene by PCR, and prepare a mixture: 1 μL of 10×PCR reaction buffer, 0.2 μL of 10 mmol/L dNTP, 0.2 μL of TaqDNA polymerase, 0.02 μL of 10 pmol/L upstream primer μL, 0.1 μL of 10 pmol/L downstream primer, 1 μL of 10×LC Green PLUS saturated fluorescent dye, 0.1 μL of probe, 1 μL of genomic DNA, add deionized water to 10 μL. The PCR reaction conditions were pre-denaturation at 95°C for 5 min, denaturation at 95°C for 1 min, annealing at 54°C for 30 s, extension at 72°C for 6 s, a total of 45 cycles, and a total extension at 72°C for 7 min. Before high-resolution melting curve analysis, denaturation and renaturation treatment: 95°C for 30s, 25°C for 2min, 94°C for 30s, 24°C for 4min. During PCR, 20 μl of paraffin oil was added to each system to prevent the liquid from volatilizing.

2) Genotype determination: transfer the PCR product into a special 96-well plate for HRM, perform HRM analysis on the Light scanner TMHR-I96, analyze the collected curve with Light Scanner Call IT software, and determine the genotype according to the difference of the melting curve .

Using unlabeled but C3-blocked probes at the 3' end, LCgreenplus DNA-binding dyes can bind in the duplex formed between the probe and the paired target strand, where one of the DNA strands (the one bound to the probe) is over-amplified If the SNPs are homozygous, the target chain has only one form, but wild homozygous and mutant homozygous have different degrees of binding to the probe, and each forms a single peak when unzipping; if the SNPs If it is heterozygous, the amplified target strand has two forms, and the two forms of probe binding determine the formation of double peaks when unzipping, thereby distinguishing three different genotypes.

Use of GPRC6A gene intron mononucleotide polymorphism site rs1606365 in preparing reagents or medicines for diagnosing or treating prostate cancer.

The assay method of the present invention measures the genomic DNA derived from human beings, and the samples are not limited, such as: body fluids (blood, ascites, urine, etc.), tissue cells (such as liver tissue), and the like. Genomic DNA can be prepared by extracting and purifying these samples. Adjust the concentration of genomic DNA to make it as consistent as possible. Using genomic DNA as a template, a nucleic acid fragment containing GPRC6A intron mutation site rs1606365 can be amplified to obtain a large number of samples for determination. The sample obtained by amplifying the DNA fragment containing the GPRC6A intron mutation point is particularly suitable for use as a determination material.

In the case of gene-assisted diagnosis, the present invention is preferentially applicable to the determination of auxiliary diagnostic reagents that exist according to the type of rs1606365 mutation in the GPRC6A intron, and the auxiliary diagnostic reagent includes specific reagents as necessary components, which correspond to the method for determining the type of rs1606365 gene mutation . Appropriate specific reagents, such as DNA fragments and/or primers for PCR amplification steps, are selected according to the assay method employed.

The advantages of the present invention are: the present invention clarifies for the first time the correlation between GPRC6A intron rs1606365 polymorphism site and PCa, and provides a method and kit for predicting PCa susceptibility, which can be used for the prevention and assistance of PCa It can also be used for diagnosis and new drug development.

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that the public has a deeper understanding of the content of the invention, and is not a limitation of the present invention. All equivalent replacements in the field made according to the disclosure of the present invention belong to this invention. protection scope of the invention.

Description of drawings

Figure 1 is a schematic diagram of the GPRC6A gene structure and intron polymorphism variation sites

Figure 2 is the melting curve of GPRC6A gene intron rs1606365 site by HRM typing

Figure 3 is the sequencing map of the rs1606365 site of the GPRC6A gene intron

Detailed ways

The abbreviations used to represent reagents in the following examples are as follows:

10×PCR buffer: 10mM Tris-HCl (pH=8.3), 500mM potassium chloride (KCl), 10mM magnesium chloride (MgCl ₂ ), 0.01% (W/V) gelatin

dNTP: deoxynucleoside triphosphate

EDTA: disodium ethylenediaminetetraacetic acid

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

Example 1: Collection of blood samples and extraction of genomic DNA:

(1) All PCa patients were diagnosed by histopathology. A total of 273 unrelated PCa patients (age: 46–93 years old, average 72.3 years old) from Beijing and Tianjin were selected, and 606 age-matched controls from the same area ( Age: 58–94 years old (mean, 70.4 years old), all male, no family history of PCa, DRE negative and PSA<4ng/mL. All subjects were of Han nationality and signed written informed consent. This study was approved by the Beijing Hospital of the Ministry of Health and the Ethical Review Committee of the Institute of Gerontology of the Ministry of Health, and complied with the World Medical Association Declaration of Helsinki: Ethical Principles for Human Medical Research. (2) Human genomic DNA was prepared according to the following method. ① First add 1000 μL of erythrocyte lysate to a labeled 1.5mLEP tube, then add 400 μL of LEDTA anticoagulant blood (invert and mix 3-5 times before adding anticoagulant blood), invert and mix well, and let stand at room temperature for 10 minutes; ② Centrifuge at 13000rpm for 30 Seconds later, remove the supernatant; ③Add 480μl nucleic acid lysate to the obtained pellet, flick the tube wall, mix well, add 20μL proteinase K (dilute proteinase K 20 times with cleavage solution), invert and mix, 65 ℃ incubator for 10 minutes, (mix up and down from time to time during the period to ensure no clots); ④ Take it out and cool it down to room temperature, add 300 μL of protein precipitation solution, mix thoroughly by inverting, let stand for 10 minutes, and centrifuge at 13000rpm for 2 minutes; Transfer the supernatant to a new EP tube, add 670 μL of pre-cooled isopropanol, invert and mix thoroughly (more than 10 times), it can be seen that the linear DNA gradually forms small clumps, centrifuge at 13,000 rpm for 2 minutes; ⑥ Discard the supernatant and ensure Leave the precipitate in EP, add 670 μL of 70% ethanol, mix up and down, and centrifuge at 13000 rpm for 2 minutes; ⑦ Discard the supernatant and let the ethanol in the tube evaporate; Analyze the concentration and purity, draw part of the DNA solution as the working solution, correct the concentration to 20ng/μL, store it at 4°C for use, and store the remaining genomic DNA in a -20°C refrigerator.

Identification and identification of embodiment 2 SNP

The present invention simultaneously detects the genotype of the rs1606365 site (its allelic site is C/G) in the intron gene region of GPRC6A by using PCR-high resolution melting curve (HRM) analysis method and PCR sequencing technology.

1) Determination of PCR-HRM primers

The DNA base sequence (SEQ ID NO.1) near rs1606365 was retrieved from Genebank, and the primer design was completed under Oligo6.0 and primer5.0 software. The target fragment is located in the intron region of the GPRC6A gene, with a total length of 180bp. The sense strand F1 (+425bp--+446bp) and the antisense strand R1 (+583bp--+604bp) are determined. The specific primer sequences are as follows:

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

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

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

2) PCR-HRM reaction system and conditions

Amplify the fragment of the rs1606365 site in the intron gene region of GPRC6A by PCR. The PCR reaction system is: 10×PCR reaction buffer 1 μL, 10 mmol/L dNTP 0.2 μL, Taq DNA polymerase 0.2 μL, 10 pmol/L upstream primer 0.02 μL, 0.1 μL of 10 pmol/L downstream primer, 1 μL of 10×LC Green PLUS saturated fluorescent dye, 0.1 μL of probe, 1 μL of genomic DNA, add deionized water to 10 μL. During PCR, 20 μl of paraffin oil was added to each system to prevent the liquid from volatilizing. The PCR reaction conditions were pre-denaturation at 95°C for 5 min, denaturation at 95°C for 1 min, annealing at 54°C for 30 s, extension at 72°C for 6 s, a total of 45 cycles, and a total extension at 72°C for 7 min. Before high-resolution melting curve analysis, denaturation and renaturation treatment: 95°C for 30s, 25°C for 2min, 94°C for 30s, 24°C for 4min.

3) HRM to determine genotype

Transfer the PCR product into a 96-well plate dedicated to HRM, and perform HRM analysis on the Light scanner TMHR-I96: start at 45°C, collect the melting curve at a slope of 0.3°C/s, and end at 98°C, use Light ScannerCall IT software to collect After the curve (Figure 2) was analyzed to determine the genotype.

4) Sequencing verification

Three samples were randomly selected from the obtained individuals of different genotypes for sequencing verification. The sequencing sample was re-amplified by PCR. The sequence of the sequencing primers was: F2: 5'-AGAAGTAAGCCCAGCTCATCAT-3' (SEQ ID No.5), R2: 5'-AGGTGCCTTACTCAAATACTGA-3' (SEQ ID No.6), the amplified fragment 180 bp long. The total PCR reaction system is 30 μL, including: 3 μL of genomic DNA, 3 μL of 10×PCRBuffer, 0.6 μL of 10 mmol/L dNTP, 0.6 μL of Taq DNA polymerase (5U/μL), 0.3 μL of upstream and downstream primers (10 pmol/μL), deionized water Supplement to a total volume of 30 μL. The PCR reaction conditions were as follows: pre-denaturation at 95°C for 5 min and then entering the main cycle, denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 60 s, 35 cycles, and extension at 72°C for 7 min. The PCR product was detected by 8% polyacrylamide gel electrophoresis, and the gel imaging system was observed and sent to the Sequencing Department of Huada Gene for sequencing verification (Figure 3).

The correlation of embodiment 3 gene SNP and PCa

Statistical methods: The Hardy-Weinberg balance test was used to study the group representativeness of the samples. Using SPSS11.0 software Pearson chi-square test to calculate the distribution frequency of alleles and genotypes of GPRC6A gene intron rs1606365 polymorphic site between the PCa case group and the normal control group, and logistic regression to evaluate the risk of PCa OR Value and its 95% CI confidence interval, with P<0.05 as the standard of significance.

Results: The distribution of the genotype and allele frequency of the SNP rs1606365 polymorphic site located in the intron of the GPRC6A gene between the cases and the control group and the association analysis with PCa are shown in Table 2.

Table 1 The genotype and allele frequency of the GPRC6A gene intron SNP rs1606365 polymorphism in China

Distribution of case-control group in population and correlation analysis with PCa

Note: OR: odds ratio; CI: confidence interval. HWE: Hardy-Weinberg equilibrium.

It can be seen from Table 1 that the G allele of the rs1606365 site of the intron of the GPRC6A gene, that is, the C allele on its DNA complementary strand, has a significantly higher distribution frequency in the patient population than in the healthy normal population. The distribution frequency (46.2%vs.38.4%) has a significant difference (age-adjusted P=0.002), and the OR value of the G site is 1.39, 95%CI: 1.13-1.71; in the recessive model (G/Gvs .C/C+C/G), the genotype distribution frequency in the case group was significantly higher than that in the control group (age-adjusted P=0.002), in the dominant model (G/G+C/Gvs.C /C), the distribution frequency of its genotype in the case group was also significantly higher than that in the control group (P=0.017, OR: 1.46, 95%CI: 1.07-1.98, age-corrected P=0.035), alleles and The association analysis between genotype and PCa showed that the G allele of the rs1606365 site in the intron of the GPRC6A gene may be positively correlated with the prevalence of PCa, and may increase the risk of PCa.

Embodiment 4 detection kit

A kit for detecting PCa-related risks is prepared, including a primer pair that can amplify the rs1606365 site of the intron of the GPRC6A gene, and other PCR-HRM corresponding reagents. The kit of the present invention is used for detection of 10 people, and is stored at -20°C in the dark, and its components, contents and sources include:

10 μL 10×PCR buffer (Pharmacia),

2 μL 10mM dNTP mixture (Pharmacia),

2 μL TaqDNA polymerase (2unit/μL) (Takara),

0.2 μL F1 (SEQ ID NO.2) (10 pmol/μL),

1 μL R1 (SEQ ID NO.3) (10pmol/μL) primer, (Shanghai Sangong Synthetic)

1 μL P probe, the nucleotide sequence shown in SEQ ID NO.4, the concentration is 10 pmol/μL; (Shanghai Sangong Synthetic)

10 μL 10×LC-green PLUS saturated fluorescent dye (Idaho, USA),

63.8 μL pure water.

Validation test: Using this kit, randomly select 10 samples from PCa patients and 10 samples from the control group, and detect the polymorphism of the rs1606365 site in the GPRC6A gene intron by PCR-HRM.

one. method

1. PCR amplification: Amplify the intron fragments of the GPRC6A gene by PCR, and prepare a mixture: 1 μL of 10×PCR reaction buffer, 0.2 μL of 10 mmol/L dNTP, 0.2 μL of TaqDNA polymerase, 0.02 μL of 10 pmol/L upstream primer μL, 0.1 μL of 10 pmol/L downstream primer, 0.1 μL of probe, 1 μL of 10×LC Green PLUS saturated fluorescent dye, 1 μL of genomic DNA, add deionized water to 10 μL. The PCR reaction conditions were pre-denaturation at 95°C for 5 min, denaturation at 95°C for 1 min, annealing at 54°C for 30 s, extension at 72°C for 6 s, a total of 45 cycles, and a total extension at 72°C for 7 min. Before high-resolution melting curve analysis, denaturation and renaturation treatment: 95°C for 30s, 25°C for 2min, 94°C for 30s, 24°C for 4min. During PCR, 20 μl of paraffin oil was added to each system to prevent the liquid from volatilizing.

2. Genotype determination: transfer the PCR product into a special 96-well plate for HRM, perform HRM analysis on the Light scanner TMHR-I96, use the Light Scanner Call IT software to analyze the collected curve, and determine the genotype according to the difference in the melting curve .

Using unlabeled probes with a C3-blocked 3' end, LCgreenplus DNA-binding dyes can bind in duplexes formed between the probe and the paired target strand, where one DNA strand (the one bound to the probe) is over-amplified If the SNPs are homozygous, the target chain has only one form, but wild homozygous and mutant homozygous bind to the probe differently, each forming a single peak when unzipping; if SNPs If it is heterozygous, the amplified target strand has two forms, and the two forms of probe binding determine the formation of double peaks when unzipping, thereby distinguishing three different genotypes.

two. result:

The results showed that the genotype of the rs1606365 site in the GPRC6A gene intron of the PCa patient samples was CG or GG; the genotype of the rs1321366 site of the control group samples was CC.

Examples of the utility of the present invention:

The detection method of the rs1606365 polymorphism of the GPRC6A gene intron of the present invention can be used to analyze the G allele site of this site on the human genome DNA, that is, the C allele site on its DNA complementary strand, and is applied to the The auxiliary diagnosis of PCa can assess the individual's risk of PCa, so as to facilitate the early prevention and treatment of PCa.

Utilizing the present invention to describe the base variation of the rs1606365 site of the intron of the GPRC6A gene, as one of the biomarkers, it can be used as a molecular target screening for drug design to help find active molecules that regulate the expression of the GPRC6A gene and promote PCa new medical research.

The nucleic acid sequence for detecting the rs1606365 site polymorphism of the intron of the GPRC6A gene established by the invention can be applied to a kit for assisting diagnosis of the PCa gene with high sensitivity and specificity.

As mentioned above, it was concluded that the polymorphism of the rs1606365 site in the intron of the GPRC6A gene was significantly associated with PCa. Therefore, the determination of this polymorphism according to the present invention can be used for gene-assisted diagnosis of PCa.

The present invention describes the new mutation site associated with GPRC6A gene intron PCa, and provides a method for determining gene polymorphism, and, according to the present invention, only a small amount of DNA samples are enough to determine the GPRC6A gene intron rs1606365 polymorphism.

The invention provides a gene-aided diagnosis method for determining PCa-related gene polymorphism.

Claims (1)

1. A test kit for detecting prostate cancer susceptibility genes, characterized in that it is made up of the following reagents: 10 μL 10×PCR buffer; 2μL 10mM dNTP mixture; 2μL Taq DNA polymerase, 2unit/μL; 0.2μL F1 primer, which is the nucleotide sequence shown in SEQ ID No.2, the concentration is 10pmol/μL; 1 μL R1 primer, the nucleotide sequence shown in SEQ ID No.3, the concentration is 10pmol/μL; 1 μL P probe, which is the nucleotide sequence shown in SEQ ID NO.4, the concentration is 10 pmol/μL; 10μL 10×LC-green PLUS saturated fluorescent dye; 63.8 μL pure water.