High resolution melting (HRM) typing primer for detecting rs 909253 locus and application of HRM typing primer

A technology for typing primers and loci, applied in the field of molecular biology, can solve problems such as difficult identification of homozygous sequence mutations, and achieve the effects of low cost, good specificity and accurate detection

Inactive Publication Date: 2020-04-03
CENT HOSPITAL XUHUI DISTRICT SHANGHAI CITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For most of the current mutation analysis methods, very Difficult to identify homozygous sequence mutations

Method used

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  • High resolution melting (HRM) typing primer for detecting rs 909253 locus and application of HRM typing primer
  • High resolution melting (HRM) typing primer for detecting rs 909253 locus and application of HRM typing primer
  • High resolution melting (HRM) typing primer for detecting rs 909253 locus and application of HRM typing primer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: HRM typing primers for detecting rs909253 locus

[0029] The present invention provides an HRM typing primer composition for detecting the rs909253 site, which consists of primer F1 and primer R1:

[0030] Preferably, the nucleotide sequence of the primer F1 is shown in SEQ ID NO.1; the nucleotide sequence of the primer R1 is shown in SEQ ID NO.2.

[0031] The primer F1 and the primer R1 are used for detecting the single nucleotide polymorphism at the rs909253 site by HRM typing.

[0032] At the same time, the present invention also provides an HRM typing reagent composition for detecting the rs909253 site;

[0033] Described HRM typing reagent is made up of primer, dNTPs, qRT-PCR amplification buffer and DNA polymerase;

[0034] The primers are composed of primer F1 and primer R1;

[0035] The invention provides an HRM typing reagent composition for detecting rs909253 site, the dNTPs are composed of fluorescein-labeled dGTP, fluorescein-labeled dATP, fluo...

Embodiment 2

[0039] Example 2: HRM typing primers for detecting rs909253 site

[0040] 1. Extraction of Genomic DNA from Whole Blood

[0041] Collect 1 mL of peripheral blood from the cubital vein of each subject, and use the DP304 kit produced by Tiangen Biochemical Technology (Beijing) Co., Ltd. for DNA extraction.

[0042] (1) Draw 300 μL of fresh blood into a 1.5mL centrifuge tube, add 900 μL of erythrocyte lysate, invert and mix well, place at room temperature for 5 minutes, during which time invert and mix several times, centrifuge at 10000rpm for 1min, absorb the supernatant, leave the white blood cell precipitate, add 200 μL buffer GA, shake until thoroughly mixed.

[0043] (2) Add 20 μL of Proteinase K solution and mix well.

[0044] (3) Add 200 μL buffer GB, mix thoroughly by inversion, place at 70°C for 10 minutes, the solution should become clear, and briefly centrifuge to remove water droplets on the inner wall of the tube cap.

[0045] (4) Add 300 μL of absolute ethanol, s...

Embodiment 3

[0061] Example 3: Verification of the detection effect of the HRM typing primers used to detect the rs909253 site

[0062] The conventional rs909253 site amplification primers were used for amplification, and the length of the amplified fragment by direct sequencing of PCR products was 149 bp. The primers were synthesized by Beijing Dingguo Biotechnology Engineering Co., Ltd. The total PCR reaction system is 30 μL, and the reaction solution is: PCR Master Mix 15 μL, template DNA 1 μL, upstream and downstream primers 1 μL (10 μM / μL), add ddH 2 0 to 30 μ L, amplified on the PTC-100 PCR amplification instrument. Reaction program: pre-denaturation at 95°C for 5 min, cycle, denaturation at 95°C for 30 s, annealing at 62°C for 45 s, extension at 72°C for 30 s, a total of 35 cycles; extension at 72°C for 5 min, storage at 4°C. The amplified product was sequenced by Beijing Dingguo Biotechnology Engineering Co., Ltd. for the target fragment (reverse sequencing), and the corresponding...

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Abstract

The invention particularly provides a high resolution melting (HRM) typing primer for detecting an rs 909253 locus and application of the HRM typing primer. The primer has the advantages of high sensitivity, good specificity and the like; and the coincidence rate between HRM results and PCR product direct-sequencing verification results is 100%. According to the provided HRM typing primer for thers 909253 locus and application of the HRM typing primer in a related reagent and detection kit for detecting mutation of the rs 909253 locus, the HRM typing primer can be used to accurately detect the rs 909253 locus, and the method has important significance and values for correlated research on the human rs 909253 locus.

Description

technical field [0001] The invention relates to the field of molecular biology, to medicine and biotechnology, in particular to HRM typing primers for detecting rs909253 sites and applications thereof. Background technique [0002] The human rs909253 site is located in the TNF-β gene. TNF-α is a multifunctional cytokine that plays an important role in promoting inflammatory response, cell proliferation and inducing apoptosis. Existing research results show that the mutation of rs909253 is closely related to the occurrence of many diseases. The single nucleotide polymorphism of rs909253 is associated with the genetic susceptibility of gastric cancer, ischemic stroke, childhood malignant lymphoma, schizophrenia, The progression of chronic obstructive pulmonary disease and the onset of neuromyelitis optica spectrum disorders are closely related. It can be seen that the rs909253 site mutation is not only related to disease susceptibility and disease symptoms, but may also becom...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/6886C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6883C12Q1/6886C12Q2600/156C12Q2527/107
Inventor 吴秀娟赵宗峰张双双陈辰陈亚琳胡程
Owner CENT HOSPITAL XUHUI DISTRICT SHANGHAI CITY
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