CN111635944A - Specific primer, kit and PCR method for detecting liver cancer susceptibility locus rs73613962 - Google Patents
Specific primer, kit and PCR method for detecting liver cancer susceptibility locus rs73613962 Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q2600/118—Prognosis of disease development
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a specific primer, a kit and a PCR method for detecting a liver cancer susceptibility locus rs73613962. The invention discovers a liver cancer susceptibility locus rs73613962, which is positioned in an intron region of a gene PRMT7, wherein a partial base sequence of the gene PRMT7 is shown as SEQ ID NO. 1, the base sequence is provided by a UCSC Genome Browser Gateway database, and the website is http:// Genome. uscs.edu/; specific primers, a kit and a PCR method for detecting the liver cancer susceptibility locus rs73613962 are developed according to a partial base sequence of the gene PRMT7, and a new approach is provided for diagnosis and prognosis of liver cancer.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a specific primer, a kit and a PCR method for detecting a liver cancer susceptibility locus rs73613962.
Background
Liver cancer is the seventh most common malignant tumor in the world, including primary liver cancer and metastatic liver cancer, the most of liver cancer is primary liver cancer, chronic hepatitis b and chronic hepatitis c are main risk factors of liver cancer, there are many risk factors for hepatitis b to develop into liver cancer, wherein the genetic factor is an important risk factor for hepatitis b to develop into liver cancer, and one of the genetic material bases causing differences among individuals is SNP. An SNP refers to a DNA sequence polymorphism caused by a variation of a single nucleotide at the genomic level, which is the most common one of human heritable genomic variations, accounting for more than 90% of all known polymorphisms. The SNP exists widely in human genome, 1 base pair exists in 500-1000 base pairs on average, the total number of the SNP is estimated to be 300 ten thousand or more, and at present, the SNP is an important basis for researching the genetic variation of common diseases of human.
The research of the prior art shows that the occurrence of liver cancer is related to genes such as EPHX1, GSTM1, GSTT1, CYP1A1, STAT4, MICA, HLA-DQ and the like, and a detection method of polymorphic sites on the genes is respectively developed.
The invention provides a Single Nucleotide Polymorphism (SNP) site related to liver cancer susceptibility and application thereof at 11/27/2012 of the university of Reddingdan of the applicant, firstly provides a partial sequence of STAT4 gene and a sequence of SNP site rs7574865 therein, and also provides a method for detecting the SNP site, a specific nucleic acid primer and a UEP primer, and also relates to a detection kit of the SNP site rs7574865 and application thereof. The rs7574865 locus is genotyped according to the invention, the method is simple, convenient and quick, the result is accurate and clear, and a new approach is provided for susceptibility identification, prevention and treatment of liver cancer.
The applicant Zhejiang loyi biomedical science and technology limited company provides an invention patent application on 2012/10/23, and discloses a kit for detecting liver cancer related risk genes, which comprises a specific primer pair and a specific fluorescent probe pair for simultaneously detecting the SNP site of rs2596542 number on an MICA gene and the SNP site of rs9275572 number on an HLA-DQ gene, a conventional component for fluorescent quantitative PCR detection and the like. The kit of the invention evaluates the risk of the individual suffering from liver cancer by simultaneously detecting the single nucleotide polymorphism site genotypes of MICA and HLA-DQ closely related to the liver cancer.
The invention provides an invention patent application at 6/4/2014 by the applicant of the university of double denier, discloses a single nucleotide polymorphism Site (SNP) related to liver cancer susceptibility and an application thereof, provides a gene partial sequence of a genetic region 6p21.3 and a sequence of one SNP site rs9275319, further provides a method for detecting the SNP site, a specific nucleic acid primer and a UEP primer, and further relates to a detection kit of the SNP site rs9275319 and an application thereof. The invention can carry out genotyping on the rs9275319 locus, has simple and quick method and accurate and clear result, and provides a new approach for susceptibility identification, prevention and treatment of liver cancer.
The applicant decoded (Shanghai) biological medicine science and technology Co., Ltd, proposed an invention patent application in 2012, 2 month 1, and discloses a noninvasive detection kit for detecting liver cancer susceptibility genes, which comprises specific primers and DNA sequencing primers, a PCR reaction system, a PCR product purification system, a DNA sequencing reaction system and the like for detecting single nucleotide polymorphism sites Tyr113His (rs1051740) on EPHX1 gene, whether GSTM1 gene is deleted (Null/Present), whether GSTT1 gene is deleted (Null/Present), and single nucleotide polymorphism sites Ile462Val (rs1048943) genotype on CYP1A1 gene. The kit of the invention evaluates the susceptibility of the liver cancer of Chinese people by detecting the genotypes of 4 mononucleotide polymorphism sites closely related to the occurrence of the liver cancer.
The above patent applications of the invention all detect polymorphic sites on genes such as EPHX1, GSTM1, GSTT1, CYP1A1, STAT4, MICA, HLA-DQ, etc., which are closely related to liver cancer, so as to judge the susceptibility of liver cancer.
The technical problems to be solved by the invention are as follows: a new liver cancer susceptibility site is discovered and detected to provide a new way for the diagnosis and prognosis of liver cancer.
Disclosure of Invention
The invention discovers a liver cancer susceptibility locus rs73613962 which is positioned in an intron region of a gene PRMT7, provides a partial base sequence of the gene PRMT7, develops a specific primer, a kit and a detection method for detecting the locus, and provides a new way for diagnosis and prognosis of liver cancer.
The invention aims to provide a specific primer for detecting a liver cancer susceptibility locus rs 73613962;
the invention also aims to provide a kit for detecting the liver cancer susceptibility locus rs 73613962;
another object of the present invention is to provide a method for performing PCR reaction using the primers as described above;
the invention also aims to provide a detection method for detecting the liver cancer susceptibility locus rs73613962, and the invention discovers a liver cancer susceptibility locus rs73613962, the locus is positioned in an intron region of a gene PRMT7, a part of base sequence of the gene PRMT7 is shown in SEQ ID NO:1, the base sequence is provided by a UCSC Genome Browser Gateway database, and the website is http:// genome.uscs.eu/; specific primers, a kit and a detection method for detecting the liver cancer susceptibility locus rs73613962 are developed according to a partial base sequence of the gene PRMT7, and a new approach is provided for diagnosis and prognosis of liver cancer. Partial nucleotide sequence of gene PRMT7
CAAAACCCCGCCTCTACAAAAAGTAGCTGGGCATGGTGATCTACGCCTGTAGTCCCAGCTACTTGGGAGATTGAGGTGGGAAATTGCTTGAGCCTGGGAGGTGGAAGCTGCAGTGATCATGCCACTGGACTCCAGCCTGGACAATAGAGTGAGACCCTTCTCAAAAAAAAAAAAAAGAAAAATTAAATGGAAAATTCCAGAAATTATTTATAAGTTGTAAGTTGTGTACCATTCTGAGTATTATTACATAATTGTTCTATTTTTAGTTATTGTTAATTTATAAATTATTATTTATATTTATTATTGTGCCTAATTTATAAATTAAACCTGGTCATAGGTAAGCTTGGAGGCTGAAGCAGCTCCACTGTGAATGCTAATCCTCCACGCTGACTTCTGACCAACCCCAGCTTCAGGAATGCCTCTAAAATTTCCGCTCTCATGCACTTTGATGTAAATCCTGCCCTCAGGTCAGAACAACTTTGATGTTATCATAAATACATACTTACCGTAAATCCTGCCCTTCGTCAAATTCCCTGTGGTATGTAAGCCCTGCGTCTGAGCCATAACAGTGTGGGGGCCCAGTTTCTCACTGCCCGAGACAAGGCTTCTGTTT(SEQ ID NO:1)。
The technical scheme of the invention is as follows:
a specific primer for detecting a liver cancer susceptibility locus rs73613962 is disclosed, wherein the nucleotide sequence of the primer is as follows:
rs73613962.F CAAAACCCCGCCTCTACAAA(SEQ ID NO:2)
rs73613962.R AAACAGAAGCCTTGTCTCGG(SEQ ID NO:3)。
a kit for detecting liver cancer susceptibility locus rs73613962 comprises the primer.
Meanwhile, the invention also provides a method for carrying out PCR reaction by using the primer, which comprises the following steps:
(1) extracting DNA of a sample to be detected;
(2) carrying out PCR reaction by taking the DNA of the sample to be detected extracted in the step (1) as a template;
the PCR reaction system is as follows: 10 mu L of KAPA HiFi buffer solution, 4 mu L of DNTPs with the concentration of 2mM, 0.6 mu L of primer rs73613962.F with the concentration of 10 mu M, 0.6 mu L of primer rs73613962.R with the concentration of 10 mu M, 2 mu L of sample DNA to be detected, 0.4 mu L of KAPA HiFi high-fidelity hot start DNA polymerase and 2.4 mu L of sterilized double distilled water.
In the method for PCR reaction by using the primers, the step of extracting the DNA of the sample to be detected refers to the step of extracting genomic DNA from human blood by using a QIAampblood Mini Kit250 Kit, and storing the extracted DNA at-20 ℃.
In the method for performing PCR reaction with the primers, the PCR reaction process is as follows: denaturation at 95 ℃ for 2 min; denaturation at 98 ℃ for 15 seconds, annealing at 60 ℃ for 30 seconds, extension at 68 ℃ for 1 minute, and circulation for 30 times; finally, it was kept at4 ℃.
In the method for performing PCR reaction by using the primers, the PCR reaction process is performed in an ABI PCR instrument ABIVeri 96-hole gradient PCR instrument.
In addition, the invention also provides a detection method for detecting the liver cancer susceptibility locus rs73613962 by using the primer, which comprises the following steps:
(1) extracting DNA of a sample to be detected;
(2) carrying out PCR reaction by taking the DNA of the sample to be detected extracted in the step (1) as a template;
(3) sequencing the PCR reaction product in the step (2);
(4) and (4) analyzing results: and (3) judging whether the subject is a liver cancer susceptible population or not by observing the base type of the subject at the rs73613962 site.
Further, the step of extracting the DNA of the sample to be detected refers to the step of extracting genomic DNA from human Blood by using a QIAamp Blood Mini Kit250 Kit, and storing the extracted DNA at-20 ℃.
Further, the PCR reaction system is as follows: 10 mu L of 2 times KAPA HiFi buffer solution, 4 mu L of DNTPs with the concentration of 2mM, 0.6 mu L of primer rs73613962.F with the concentration of 10 mu M, 0.6 mu L of primer rs73613962.R0.6 mu L with the concentration of 10 mu M, 2 mu L of sample DNA to be tested, 0.4 mu L of KAPA HiFi high-fidelity hot start DNA polymerase and 2.4 mu L of sterilized double distilled water.
Further, the PCR reaction process comprises: denaturation at 95 ℃ for 2 min; denaturation at 98 ℃ for 15 seconds, annealing at 60 ℃ for 30 seconds, extension at 68 ℃ for 1 minute, and circulation for 30 times; finally, it was kept at4 ℃.
Further, the judging method in the step (4) is as follows: if the base of the subject at the rs73613962 site is T, the subject is a non-liver cancer susceptible population; if the base of the subject at the rs73613962 site is G, the subject is the liver cancer susceptible population.
The invention has the following beneficial effects:
the invention discovers a liver cancer susceptibility locus rs73613962 which is positioned in an intron region of a gene PRMT7 and provides a partial base sequence of the gene PRMT7, develops a specific primer, a kit and a detection method for detecting the liver cancer susceptibility locus rs73613962 according to the partial base sequence of the gene PRMT7, and provides a new way for diagnosis and prognosis of liver cancer.
Drawings
FIG. 1 is a base sequence and a base sequence chart of a PCR reaction product of genomic DNA of a subject 1 in example 1, in which bases of the rs73613962 site are marked with gray boxes;
FIG. 2 is a base sequence and a base sequence chart of the PCR reaction product of the genomic DNA of the subject 2 in example 2, in which bases of the rs73613962 site are marked with gray boxes.
Detailed Description
The technical solution of the present invention will be described in further detail with reference to the following embodiments, but the present invention is not limited thereto.
The starting materials used in the following examples are all available from conventional commercial sources unless otherwise specified.
Example 1
According to a partial base sequence SEQ ID NO 1 of a gene PRMT7 provided by a UCSC Genome Browser Gateway database with the website address of http:// Genome. uscs. edu/, and according to a liver cancer susceptibility locus rs73613962 positioned in an intron region of the gene PRMT7, a Primer base sequence for detecting the liver cancer susceptibility locus rs73613962 is designed by an online Primer design tool Primer 3, and the designed Primer base sequence is synthesized by a biological engineering (Shanghai) company Limited to obtain the following specific primers:
rs73613962.F CAAAACCCCGCCTCTACAAA(SEQ ID NO:2)
rs73613962.R AAACAGAAGCCTTGTCTCGG(SEQ ID NO:3)。
the kit for detecting the liver cancer susceptibility locus rs73613962, namely a KAPAHiFi high-fidelity hot start DNA polymerase PCR reaction kit, is prepared by using the specific primers.
The detection method for detecting the liver cancer susceptibility locus rs73613962 of the genome DNA of the subject 1 by using the primer comprises the following steps:
(1) 2-3 mL of peripheral venous Blood of the subject 1 was extracted, and then genomic DNA of the subject 1 was extracted using a QIAamp Blood Mini Kit250 (purchased from QIAGEN) Kit by the following method and stored in a freezer at-20 ℃ for future use;
A. pipetting 20. mu.L of proteinase K into a new 1.5ml EP tube;
B. aspirating 200 μ L of blood sample into the EP tube;
C. sucking 200 mu L of Buffer AL into the sample, shaking the sample by vortex for 15s, mixing the sample uniformly, and carrying out water bath at 56 ℃ for 10 min;
D. the centrifugal machine rapidly centrifugalizes for a short time, and liquid drops on the 1.5ml EP tube cover and the tube wall are gathered to the tube bottom;
E. taking absolute ethyl alcohol (about 200 mu L) with the same volume as the sample, shaking for 15s by a vortex oscillator, and then rapidly centrifuging by using a centrifugal machine;
F. transferring the centrifuged solution into an adsorption column provided by the kit (the adsorption column is placed in a 2ml centrifuge tube), centrifuging at 8000rpm for 1min, discarding the centrifuge tube and the filtrate in the tube, and placing the adsorption column into a new 2ml centrifuge tube;
G. opening the tube cover of the adsorption column, adding 500 μ L Buffer AW1, centrifuging at 8000rpm for 1min, discarding the centrifuge tube and filtrate, and placing the adsorption column into a new 2ml centrifuge tube;
H. opening the tube cover of the adsorption column, adding 500 μ L Buffer AW2, centrifuging at 12000rpm for 3min, discarding the filtrate, placing back in the centrifuge, and centrifuging at 12000rpm for 1 min.
I. Transferring the adsorption column to a new 1.5ml centrifuge tube, adding sterilized double distilled water which is warm-bathed at 65 ℃, incubating for 1min at room temperature, placing the tube in a centrifuge, centrifuging for 1min at 8000rpm, discarding the adsorption column, and measuring the concentration and purity of the extracted DNA by using a Nano Drop spectrophotometer to determine that the DNA meets the extraction standard (A260/A280> 1.8).
(2) Configuring a PCR reaction system according to KAPA HiFi high-fidelity hot start DNA polymerase, adding the specific primer for detecting the liver cancer susceptibility site rs79613962 and the genome DNA of the subject 1 into the system, wherein the PCR reaction system is shown in the following table 1:
TABLE 1 PCR reaction System
It should be noted that: in this example, mM, μ M represent millimoles per liter and micromoles per liter;
and (3) PCR reaction process:
the reaction program was set on an ABI PCR instrument ABI Veriti 96-well gradient PCR instrument (purchased from ABI corporation): 2 minutes at 95 ℃; 30 cycles: 15 seconds at 98 ℃, 30 seconds at 60 ℃ and 1 minute at 68 ℃; infinity at4 ℃;
(3) sending the PCR reaction product to a company Limited in Biotechnology engineering (Shanghai) to perform Sanger sequencing, wherein the sequencing result returned by the company is the base sequence and the base sequence diagram of the product;
(4) and (4) analyzing results: judging whether the subject 1 is a liver cancer susceptible population or not by observing the base type of the subject 1 at the rs73613962 site; if the base of the subject 1 at the rs73613962 site is T, the non-liver cancer susceptible population is obtained; if the base of the subject 1 at the rs73613962 site is G, the liver cancer susceptible population is obtained.
The base sequence and base sequence diagram of the PCR reaction product of the genomic DNA of the subject 1 in this example are shown in FIG. 1, wherein the base of the rs73613962 site is marked by a gray frame, and the result of the reverse complementary base sequence is determined, so that the base of the rs73613962 site is A.
Example 2
The detection method and the detection flow of the liver cancer susceptibility locus rs73613962 for detecting the genome DNA of the subject 2 are the same as those in the embodiment 1,
the base sequence and base sequence chart of the PCR reaction product of the genomic DNA of the subject 2 in this example are shown in FIG. 2, wherein the base of the rs73613962 site is marked by a gray frame, and the result of the reverse complementary base sequence is determined, so that the base of the rs73613962 site is C.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
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Claims (6)
1. A specific primer for detecting a liver cancer susceptibility locus rs73613962 is characterized in that the nucleotide sequence of the primer is as follows:
rs73613962.F CAAAACCCCGCCTCTACAAA(SEQ ID NO:2)
rs73613962.R AAACAGAAGCCTTGTCTCGG(SEQ ID NO:3)。
2. a kit for detecting liver cancer susceptibility locus rs73613962, which comprises the primer of claim 1.
3. A method for performing a PCR reaction using the primer of claim 1, comprising the steps of:
(1) extracting DNA of a sample to be detected;
(2) carrying out PCR reaction by taking the DNA of the sample to be detected extracted in the step (1) as a template;
the PCR reaction system is as follows: 10 mu L of KAPA HiFi buffer solution, 4 mu L of DNTPs with the concentration of 2mM, 0.6 mu L of primer rs73613962.F with the concentration of 10 mu M, 0.6 mu L of primer rs73613962.R with the concentration of 10 mu M, 2 mu L of sample DNA to be detected, 0.4 mu L of KAPAHiFi high-fidelity hot start DNA polymerase and 2.4 mu L of sterilized double distilled water.
4. The method of claim 3, wherein the extracting of the DNA from the sample to be tested is performed by extracting genomic DNA from human blood using QIAampblood Mini Kit250 Kit, and storing the extracted DNA at-20 ℃.
5. The method of claim 3, wherein the PCR reaction scheme is: denaturation at 95 ℃ for 2 min; denaturation at 98 ℃ for 15 seconds, annealing at 60 ℃ for 30 seconds, extension at 68 ℃ for 1 minute, and circulation for 30 times; finally, it was kept at4 ℃.
6. The method of claim 3, wherein the PCR reaction protocol is performed in an ABI PCR instrument ABIVeriti 96-well gradient PCR instrument.
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