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Primer and method for detecting HLA-DQ gene rs9275319 site polymorphism

A technology of HLA-DQ and site polymorphism, which is applied in biochemical equipment and methods, recombinant DNA technology, and microbial measurement/inspection, can solve the problems of high sample quality requirements, low specificity and sensitivity, and achieve High sensitivity, improved detection efficiency, and good specificity

Inactive Publication Date: 2016-08-03
CHENGDU ZHONGCHUANG QINGKE MEDICAL LAB CO LTD
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Problems solved by technology

[0005] Aiming at the above-mentioned problems existing in the prior art, the present invention provides a primer and a detection method for detecting the polymorphism of the HLA-DQ gene rs9275319 site, which can effectively solve the problem of low specificity and sensitivity and high requirements for sample quality. question

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  • Primer and method for detecting HLA-DQ gene rs9275319 site polymorphism
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  • Primer and method for detecting HLA-DQ gene rs9275319 site polymorphism

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Embodiment 1

[0024] A large number of primers were designed for the rs9275319 locus of the HLA-DQ gene. Through optimization and comparison of the primer reaction conditions, a pair of primers with good specificity was screened out. They were HLA-DQ-F: 5'-CTTCCATGAACCTTACAG-3' (SEQIDNo :1); HLA-DQ-R: 5'-AAATGGCTACTTCCCTA-3' (SEQ ID No: 2), the primer amplified fragment is located at chrl:32698036-32698748, the length is 713, R in the sequence is a mutant base, see the specific sequence (SEQIDNo: 3).

[0025] Use this primer for PCR amplification of the genomic DNA to be tested. The PCR amplification system is: aμL of DNA template to make the content 100-150ng, 19-aμL of deionized water, and the forward primer 3.0μL with a concentration of 5pmol / μL, and the concentration is 5pmol / μL reverse primer 3.0μL, PrimerSTARMAX 25.0μL; amplification reaction conditions: 95℃ pre-denaturation 5min, 95℃ denaturation 30s, 55℃ annealing 15s, 72℃ extension 15s, 30 cycles, finally 72℃ extension 5min Then stor...

Embodiment 2

[0026] Example 2 Detection of HLA-DQ gene rs9275319 polymorphism

[0027] (1) Extract DNA samples of EDTA anticoagulated peripheral blood (cubital venous blood), and the extraction method refers to the instructions of the blood genomic DNA extraction kit (purchased from Beijing Tiangen Biochemical Technology Co., Ltd.);

[0028] (2) Carry out PCR amplification on the above-mentioned DNA samples, PCR amplification adopts PrimerSTARMAX Premix (2X) (purchased from Beijing Tiangen Biochemical Technology Co., Ltd.), the reaction system is shown in Table 1, the primer concentration is 5pmol / μL, the template DNA The amount is 100-150ng, add aμL (calculated according to the extracted DNA concentration), the PCR amplification reaction conditions are shown in Table 2; PCR products are separated by 2% agarose gel electrophoresis, and DNA gel recovery kit is used to recover DNA; electrophoresis parameters It is: voltage 120V, 400mA, time 30min; the glue recovery method refers to the instructio...

Embodiment 3

[0040] Example 3 Detecting the specificity of SNP sites of susceptibility genes for liver cancer

[0041] This detection method defines specificity as the phenomenon of no set peaks, background peaks, clutter peaks, and floating peaks in the peak graph of the sequencing results.

[0042] According to the detection method provided by the present invention, 50 samples are detected. The sequencing peak pattern is single, without double peaks, background peaks and other phenomena. The detection results of 50 samples are all the same. See the peak pattern results. figure 2 , Indicating that the specificity of the detection method provided by the present invention is 100%.

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Abstract

The invention provides a primer for detecting the HLA-DQ gene rs9275319 site polymorphism .The primer includes a forward primer 5'-CTTCCATGAACCTTACAG-3' and a reverse primer 5'-AAATGGCTACTTCCCTA-3' .A method for detecting the HLA-DQ gene rs9275319 site polymorphism with the primer includes the following steps that 1, samples are collected, and genome DNA of the samples is extracted; 2, the genome DNA is subjected to PCR amplification through the primer, and the amplification products are subjected to gel recovery; 3, the gel recovery products are subjected to concentration measurement and PCR amplification and purified; 4, the purified products in the step 3 are subjected to sample loading in a 3730-type full-automatic sequence analysis meter, and SNP typing is analyzed with Chromas software .The primer and the detection method are good in specificity, high in sensitivity and good in accuracy.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a primer and a detection method for detecting the polymorphism of the rs9275319 locus of the HLA-DQ gene. Background technique [0002] Liver cancer is a common primary liver malignant tumor. It is a common malignant tumor in my country. Since the end of the last century, it has rapidly risen to the second place in my country's malignant tumors, second only to lung cancer. The average annual survival rate of liver cancer is only 9%. The number of deaths from liver cancer in my country each year accounts for 45% of the total global liver cancer deaths, which brings a serious burden to the patients' families and society. my country is an area with a high incidence of hepatitis B. Only HBsAg carriers account for 34.3% (350 million) of the global carriers. According to investigations, the incidence of liver cancer among carriers of hepatitis B virus (HBV) in my country is relative...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6886C12Q2600/156
Inventor 邓银张蓉陈思翔李江林
Owner CHENGDU ZHONGCHUANG QINGKE MEDICAL LAB CO LTD
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