Probe and method for detecting ESR2 gene polymorphism

A gene polymorphism and probe technology, which is applied in biochemical equipment and methods, microbial measurement/testing, DNA/RNA fragments, etc., can solve the problems of high cost, inapplicability of large sample detection, time-consuming and labor-intensive, etc., to achieve The effect of high sensitivity, clear genotyping, and simple method operation

Inactive Publication Date: 2020-08-07
上海利康精准医疗技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the commonly used ESR2 gene mutation detection method is direct DNA sequencing method. PCR products can be directly analyzed by DNA se

Method used

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  • Probe and method for detecting ESR2 gene polymorphism
  • Probe and method for detecting ESR2 gene polymorphism
  • Probe and method for detecting ESR2 gene polymorphism

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] 1. Construct and prepare the wild-type standard plasmid and mutant standard plasmid containing the target gene locus. The accuracy of the sequence can be confirmed by enzyme digestion and sequencing. The genotype of the wild-type standard plasmid rs1256054 is GG; the genotype of the mutant standard plasmid rs1256054 is CC. The standard plasmid DNA concentration was normalized to 10 ng / μL.

[0036] 2. Use the online software Primer 3 to design primers to strengthen the complementary sequence of the primer loop and the target DNA sequence. The sequence of the forward primer is: 5′-GCAACGGGTCACGTACGCAA TAGGGATGAGGGGAAATGCG-3′; the sequence of the reverse primer is: 5′-GCAACGGGTCACGTACGCAA CCCGATAAAACATGGCCCAG-3 '; the sequence of the probe is 5'-FAM-CGAGAGTTAAAACTCCAACACAAAGAATATCTCG-BHQ1-3'.

[0037] 3. Reaction system optimization:

[0038] 1) Probe volume: set probe volumes of 0.01 μL, 0.05 μL, 0.1 μL and 0.5 μL respectively, and keep other conditions unchanged, and p...

Embodiment 2

[0045] 1. Using the silica gel adsorption method to extract the genomic DNA of the oral epithelial cells of a test subject, the concentration and purity of the DNA were detected by electrophoresis gel imaging, and the concentration of the sample to be tested was standardized to 10 ng / μL.

[0046] 2. The detection method is as follows: add 7.5 μL of PCR Mix, 0.3 μL of forward primer solution, 0.3 μL of reverse primer solution, and 0.1 μL of probe to the PCR reaction wells in sequence. Add 2 μL of DNA to each reaction well, make up 15 μL with sterilized double distilled water; carry out the reaction on the fluorescent quantitative PCR detection system SLAN-96P, the PCR reaction conditions are 95°C pre-denaturation for 5 minutes; 95°C denaturation for 30 seconds, 60°C annealing for 30 seconds, Extend at 72°C for 30 seconds, 45 cycles; extend at 72°C for 10 minutes; denature at 95°C for 1 minute, anneal at 40°C for 1 minute, monitor the fluorescence signal in real time during the p...

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Abstract

The invention discloses a method for detecting ESR2 gene polymorphism. The method comprises the following steps: determining an ESR2 gene mutation site; designing and synthesizing a probe and a primeraccording to the mutation site; the method comprises the following steps: mixing a detected ESR2 target gene, a probe, a primer and an enzyme, carrying out fluorescent quantitative PCR, and monitoring a fluorescence signal; calculating a Tm value displayed by the hybridization peak, and judging the type of the ESR2 gene according to the Tm value; the invention also discloses a probe for detectingthe polymorphism of the ESR2 gene. The nucleotide sequence of the probe comprises SEQ ID NO. 3; the invention provides the probe and the method which are simple to operate and can be used for quicklydetecting the polymorphism of the ESR2 gene.

Description

technical field [0001] The invention relates to a probe and method for detecting ESR2 gene polymorphism. Background technique [0002] Breast cancer is the huge "number one killer" of women's health, with 500,000 women dying of this cancer every year around the world. This disease great majority occurs in 40-60 years old, the women before and after menopause. The incidence of breast cancer worldwide is increasing at an annual rate of 0.2%-3%. In 2012, the number of new breast cancer cases was 1.7 million. In Western developed countries such as Europe and the United States, breast cancer has become one of the main causes of death for women, and one out of every 8-10 women will suffer from breast cancer in their lifetime. In my country, the incidence rate of breast cancer is also increasing year by year, reaching 42.55 / 100,000. In Beijing, Shanghai, Tianjin and other big cities, breast cancer has become the first female malignant tumor incidence. The occurrence of sporadic ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12N15/11C12Q1/6858
CPCC12Q1/6858C12Q1/6886C12Q2600/156C12Q2531/113C12Q2563/107
Inventor 傅咏南毛丹丹黄芬芬张奕
Owner 上海利康精准医疗技术有限公司
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