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Real-time fluorescence PCR method for detecting rs1800497 site of ANNK1 gene and primer probe combination thereof

A primer-probe, real-time fluorescence technology, used in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve problems such as specificity and sensitivity difficulties, and achieve high sensitivity, simple operation, and simple result analysis. Effect

Pending Publication Date: 2020-04-03
SHANXI LIFEGEN
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  • Application Information

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Problems solved by technology

For those skilled in the field of gene detection, the current application of real-time fluorescent PCR based on TaqMan probes is mainly due to difficulties in specificity and sensitivity; because it is not simply to combine primers with probes, but to target ANKK1 A set of specific primers and TaqMan probes were designed for the rs1800497 site to efficiently, quickly and accurately detect the genotype of the sample to be tested

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  • Real-time fluorescence PCR method for detecting rs1800497 site of ANNK1 gene and primer probe combination thereof
  • Real-time fluorescence PCR method for detecting rs1800497 site of ANNK1 gene and primer probe combination thereof
  • Real-time fluorescence PCR method for detecting rs1800497 site of ANNK1 gene and primer probe combination thereof

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specific Embodiment

[0044] Specific embodiment: TaqMan probe real-time fluorescent PCR method detects the allele of rs1800497 site.

[0045] 1. Extraction and dilution of DNA samples

[0046] After collecting venous blood in vacuum blood collection tubes anticoagulated with ethylenediaminetetraacetic acid (EDTA) according to conventional methods, DNA was extracted using the QIAamp DNA Mini Blood Kit (Qiagen, Germany) kit; the extracted DNA was concentrated using NanoDrop 2000 Determination (A260 / 280=1.95~2.15). Using the above method, respectively measure the concentration of 104 cases of Bouyei DNA samples, and then use PCR grade H 2 O Dilute the sample to 10 ng / μl.

[0047] 2. Design primers and probes

[0048] Specific upstream primers: G allele: 5’-AGCTGGGCGCCTGCCGC-3’; A allele:

[0049] 5'-AGCTGGGCGCCTGCCGT-3';

[0050] Universal downstream primer: 5'-GCAAATGTCCACGCCCGCA-3';

[0051] Probe: 5'-FAM-AGCACTTTGAGGATGGCTGTGT-BHQ2-3';

[0052] Among them, FAM is 6-carboxyfluorescein; BHQ2 ...

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Abstract

The invention belongs to the field of gene diagnosis, and discloses a real-time fluorescence PCR method for detecting a rs1800497 site of an ANKK1 gene and a primer probe combination thereof. A upstream primer FpC is FpC; 5'-AGCTGGGCGCCTGCCGC-3', wherein an upstream primer FpT is 5'-AGCTGGGCGCCTGCCGT-3', a downstream primer Rp is 5'-GCAAATGTCCACGCCCGCA-3', and a probe Probe is 5'-FAM-AGCACTTTGAGGATGGCTGTGT-BHQ2-3'. After a PCR reaction is finished, a result can be analyzed by judging whether an amplification curve exists or not and combining with a Ct value. The detection method provided by the invention is used for detecting rs1800497 site polymorphism, and has the advantages of simplicity and convenience in operation, high resolution, no pollution, high flux and the like.

Description

technical field [0001] The invention belongs to the field of pharmacogenomics and gene detection, and specifically relates to the design of specific primers and probes for the rs1800497 site (ANNK1, c.2137G>A) allele of ANNK1 gene, and a detection method. Background technique [0002] Depression is a common mental illness in modern people, which seriously affects the mental health and life safety of patients. Clinically, the main symptoms of depression include emotional loss, depression, memory loss, etc., and severe patients may commit suicide. Depressed patients may self-medicate with smoking and develop tobacco dependence. However, in a state of mental illness, some mental symptoms such as hallucinations and delusions can lead to negative emotions such as anxiety, tension and depression, which indirectly lead to an increase in the patient's smoking. The survey results show that the smoking rate of patients with schizophrenia is significantly higher (up to 80%) than t...

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/686C12N15/11
CPCC12Q1/6883C12Q1/686C12Q2600/156C12Q2600/106
Inventor 王会娟康星韩敏
Owner SHANXI LIFEGEN
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