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Primer and method for detecting CYP2D6 gene polymorphism

A gene polymorphism and amplification primer technology is applied in the field of primers for detecting CYP2D6 gene polymorphism, achieving the effects of good specificity, improved therapeutic effect and reduced adverse reactions

Inactive Publication Date: 2016-04-20
GUANGZHOU KINGMED DIAGNOSTICS GRP CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have found that there are two highly homologous pseudogenes upstream of the CYP2D6 gene, called the CYP2D7P gene and the CYP2D8P gene. A thymine (T) insertion at position 226 of exon 1 alters the reading frame and terminates transcription prematurely; the CYP2D8P gene is a mutant pseudogene containing multiple breakpoints

Method used

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  • Primer and method for detecting CYP2D6 gene polymorphism
  • Primer and method for detecting CYP2D6 gene polymorphism
  • Primer and method for detecting CYP2D6 gene polymorphism

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Experimental program
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Embodiment 1

[0025] Embodiment one, primer

[0026]The primers provided by the present invention are shown in Table 1, including nested PCR amplification primers and SNaPshotPCR primers; the nested PCR amplification primers include nested A round PCR amplification primers and nested B round PCR amplification primers, so The nested round B PCR amplification primers correspond to the SNaPshotPCR primers. All primer sequences provided by the present invention are compared through UCSC database, and there is no known SNP site.

[0027] Table 1 Primers provided by the present invention

[0028]

Embodiment 2

[0029] Embodiment two, the specificity of primer

[0030] The primers provided by the present invention were blasted in UCSC, and the results were as follows:

[0031] 1) CYP2D6 gene-specific primers were used for Blast in UCSC without other homologous genes. 2) The CYP2D6_C2850T amplified fragment is located at chr22:42127836+42128150, with a length of 315bp, and the amplified sequence is as follows figure 1 ; The amplified fragment of CYP2D6_G3183A is located at chr22:42127471-42127709, the length is 239bp, and the amplified sequence is as follows figure 2 ; The CYP2D6_G4180C amplified fragment is located at chr22:42126391-42126685, with a length of 295bp, and the amplified sequence is as follows image 3 ; The CYP2D6_C100T amplified fragment is located at chr22:42130611-42130821, the length is 211bp, and the amplified sequence is as follows Figure 4 ; The CYP2D6_G1846A / CYP2D6_G1661C amplified fragment is located at chr22:42128876+42129258, the length is 383bp, and the...

Embodiment 3

[0033] Example 3, Detection of CYP2D6 Gene Polymorphism

[0034] 1) Extract DNA samples from EDTA anticoagulated peripheral blood. The extraction method refers to the instructions of TIANampBloodDNAKit (purchased from Tiagen, product number DP318), and the DNA samples are diluted to 100ng / μL for later use.

[0035] 2) Prepare the primer mixture: prepare the nested round A PCR primer mixture, CYP2D6-Fwd:CYP2D6-Rev:H2O=1:1:3, the final primer concentration is 1pmol / μL, vortex and mix well, and prepare for use after short centrifugation; preparation B 轮PCR引物混合物,将5对引物如下,CYP2D6_C2850T-Fwd / CYP2D6_C2850T-Rev,CYP2D6_C100T-Fwd / CYP2D6_C100T-Rev,CYP2D6(1661 / 1846)-Fwd / CYP2D6(1661 / 1846)-Rev,CYP2D6_G3183A-Fwd / CYP2D6_G3183A -Rev, CYP2D6_G4180C-Fwd / CYP2D6_G4180C-Rev were mixed in equal volumes, and the final concentration of primers was 0.5pmol / μL; after short centrifugation, it was ready for use; PCR amplification was performed using Q5 ? Hot start ultra-fidelity 2XMasterMix (purchased from ...

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Abstract

The invention belongs to the technical field of biological detection, and in particular relates to a primer and a method for detecting CYP2D6 gene polymorphism. The primer for detecting the CYP2D6 gene polymorphism comprises a nested PCR amplification primer and an SNaPshot PCR primer; the nested PCR amplification primer comprises a nested A-turn amplification primer and a nested B-turn amplification primer. The primer for detecting the CYP2D6 gene polymorphism has the advantages of good specificity, no cross reaction and high accuracy, detection of the CYP2D6 gene polymorphism is realized, and the primer has very important significance for realizing gene oriented individualized treatment, continuously improving treatment effects and reducing untoward effects.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a primer and a method for detecting CYP2D6 gene polymorphism. Background technique [0002] CYP2D6 is the first P450 enzyme confirmed to be controlled by a single gene. CYP2D6 is located on chromosome 22, which contains 9 exons and 8 introns, with a total length of about 7kb. It is a complete functional gene . Studies have found that there are two highly homologous pseudogenes upstream of the CYP2D6 gene, which are called CYP2D7P gene and CYP2D8P gene. A thymine (T) was inserted at the 226th position of exon 1, which changed the reading frame and caused early termination of transcription; the CYP2D8P gene is a mutant pseudogene containing multiple breakpoints. Neither CYP2D7P nor CYP2D8P genes are expressed in the human footprint, and only CYP2D6 is expressed in the liver, intestine, kidney, and human brain. [0003] Modern studies have found that the ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q1/6883C12Q2600/106C12Q2600/156C12Q2531/113C12Q2537/143C12Q2549/119C12Q2565/125
Inventor 梁耀铭于世辉赵薇薇燕启江胡昌明罗锦霞
Owner GUANGZHOU KINGMED DIAGNOSTICS GRP CO LTD
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