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Liquid phase chip method and kit for detecting polymorphism of cytochrome P450 1A2 (CYP1A2) genes

A technology of CYP1A2 and gene polymorphism, which is applied in the fields of medicine and biology, and in vitro detection technology, can solve the problems of not really satisfying clinical diagnosis detection, insufficient sensitivity, poor repeatability, etc., and achieve rapid detection, high sensitivity, and high specificity sexual effect

Inactive Publication Date: 2012-04-04
邵棠
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Among the currently widely used molecular biology methods for detecting CYP1A2 gene polymorphisms at home and abroad, fluorescent quantitative PCR has limitations in detection throughput, so none of them can really meet the needs of clinical diagnosis and detection.
The traditional solid-phase biochip (Biochip) technology has outstanding weaknesses such as poor repeatability, insufficient sensitivity, and cumbersome operations.

Method used

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  • Liquid phase chip method and kit for detecting polymorphism of cytochrome P450 1A2 (CYP1A2) genes
  • Liquid phase chip method and kit for detecting polymorphism of cytochrome P450 1A2 (CYP1A2) genes
  • Liquid phase chip method and kit for detecting polymorphism of cytochrome P450 1A2 (CYP1A2) genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Liquid-phase chip combined detection method for CYP1A2 gene polymorphism CYP1A2*1F (-163C>A)

[0041] The specific detection method includes the following steps:

[0042] 1. Preparation of microsphere mixture for detection of CYP1A2 gene polymorphism

[0043] 1. Synthesize oligonucleotide probes according to the following sequence:

[0044] CYP1A2*1F-wt: 5'-AminolinkerC12 ATGCGTCCTG(G)GCCCACAGAG-3', as shown in SEQ ID NO.1;

[0045] CYP1A2*1F-mut: 5'-AminolinkerC12 ATGCGTCCTG(T)GCCCACAGAG-3', as shown in SEQ ID NO.2;

[0046] 2. Coupling the above oligonucleotide probes containing amino modification with two kinds of carboxyl microspheres No. 11 and No. 15 respectively

[0047] 2.1 Take out a small portion of fresh dry powdered EDC stored at -20°C and equilibrate to room temperature;

[0048] 2.2 Using dH 2 O respectively dissolve the oligonucleotide probes of CYP1A2*1F-wt and CYP1A2*1F-mut at a concentration of 1mM (1nmol / μl);

[0049] 2.3 Vortex the s...

Embodiment 2

[0117] Example 2: Liquid-phase chip combined detection method for CYP1A2 gene polymorphism CYP1A2*1D(-2467delT)

[0118] The specific detection method includes the following steps:

[0119] 1. Preparation of microsphere mixture for detection of CYP1A2*1D-wt and CYP1A2*1D-mut gene polymorphisms

[0120] 1. Synthesize oligonucleotide probes according to the following sequence:

[0121] CYP1A2*1D-wt: 5'-AminolinkerC12 GTTGGGGTTCATGTGCCACAA-3', as shown in SEQ ID NO.3;

[0122] CYP1A2*1D-mut: 5'-AminolinkerC12GTTGGGGTTCTGTGCCACAA-3', such as SEQ ID NO.4

[0123] 2. Coupling the above oligonucleotide probes containing amino modifications to two kinds of carboxyl microspheres numbered 17 and 21 respectively

[0124] 2.1 Take out a small portion of fresh dry powdered EDC stored at -20°C and equilibrate to room temperature;

[0125] 2.2 with dH 2 O respectively dissolve the oligonucleotide probes of CYP1A2*1D-wt and CYP1A2*1D-mut at a concentration of 1mM (1nmol / μl);

[0126] 2....

Embodiment 3

[0194] Example 3: Liquid chip combined detection method for CYP1A2 gene polymorphisms CYP1A2*1F (-163C>A) and CYP1A2*1D (-2467delT)

[0195] The specific detection method includes the following steps:

[0196] 1. Preparation of microsphere mixture for detection of CYP1A2*1F-wt, CYP1A2*1F-mut, CYP1A2*1D-wt, CYP1A2*1D-mut genes

[0197] 1. Synthesize oligonucleotide probes according to the following sequence:

[0198] CYP1A2*1F-wt: 5'-AminolinkerC12ATGCGTCCTG(G)GCCCACAGAG-3', as shown in SEQ ID NO.1;

[0199] CYP1A2*1F-mut: 5'-AminolinkerC12ATGCGTCCTG(T)GCCCACAGAG-3', as shown in SEQ ID NO.2;

[0200] CYP1A2*1D-wt: 5'-AminolinkerC12GTTGGGGTTC(A)TGTGCCACAA-3', as shown in SEQ ID NO.3;

[0201] CYP1A2*1D-mut: 5'-AminolinkerC12GTTGGGGTTC()TGTGCCACAA-3', as shown in SEQ ID NO.4

[0202] 2. Coupling the above oligonucleotide probes containing amino modifications to the four kinds of carboxyl microspheres numbered 11, 15, 17, and 21 respectively

[0203] 2.1 Take out a small port...

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Abstract

The invention discloses a liquid phase chip detection method and a detection kit for detecting the polymorphism of cytochrome P450 1A2 (CYP1A2) genes. The method comprises the following steps of: designing primers and probes for polymorphism CYP1A2*1F (-163C>A) and CYP1A2*1D (-2467delT) of the CYP1A2 genes, hybridizing a probe-microsphere mixture formed by covalent bonding of the probes and microspheres and a polymerase chain reaction (PCR) amplification product, adding streptavidin phycoerythrin, and thus detecting fluorescence signals of different microspheres and determining the polymorphism of the CYP1A2 genes in a sample to be detected. The method and the kit have the advantages of high sensitivity, high flux, quickness and accuracy in detection, and the like, can be used for detecting and identifying the polymorphism of the CYP1A2 genes, and also provide an important reference for medicament metabolism and personalized medication.

Description

technical field [0001] The invention relates to the fields of in vitro detection technology, medicine and biotechnology, in particular to a liquid phase chip detection method and a detection kit for CYP1A2 gene polymorphism. Background technique [0002] CYP1A2 is an important cytochrome P450 enzyme (cytochrome P450, CYP), the gene is located on human chromosome 15, including 7 exons and 6 introns. CYP1A2 is involved in the biotransformation of various drugs in the body, as well as the metabolism of some environmental toxins and the activation of carcinogens. At present, it is known that CYP1A is involved in the metabolism of dozens of drugs such as caffeine, warfarin, theophylline, propranolol, mexiletine, verapal, nifedipine, and tacrine. CYP1A2 gene polymorphism is the basis for individual differences in CYP1A2 enzyme activity and inducibility, and is an important reason for individual and racial differences in drug efficacy and toxicity. Therefore, the identification a...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 邵棠涂文娟李玲邓景人陈庆
Owner 邵棠
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