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Oligonucleotide for detecting cytochrome P450 enzyme series mutation site and gene chip

A technology of oligonucleotide probes and detection probes, which is applied in the field of nucleic acid detection of DNA genotypes, can solve problems such as the inability to determine mutation sites and mutation forms, high technical requirements for operators, and difficulty in popularization, achieving remarkable practicality value, the effect of preventing side effects

Inactive Publication Date: 2007-10-17
INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The conformation-based SNP detection method is relatively simple and fast, but it cannot give sequence information, so it is impossible to determine the specific mutation site and mutation form, and cannot distinguish multiple mutations in a fragment, and it requires high technical requirements for operators, so it is difficult to popularize; The other is genotyping techniques based on fluorescently labeled hybridization reactions, including oligonucleotide ligation analysis, pyrophosphate DNA sequencing, direct heterozygote sequencing, and allele-specific primer extension, etc.
This type of method needs to invest a lot of money and purchase special instruments and probes and other supporting reagents. Only by realizing high-throughput detection and reducing the cost of use can it be widely used

Method used

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  • Oligonucleotide for detecting cytochrome P450 enzyme series mutation site and gene chip
  • Oligonucleotide for detecting cytochrome P450 enzyme series mutation site and gene chip
  • Oligonucleotide for detecting cytochrome P450 enzyme series mutation site and gene chip

Examples

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Effect test

Embodiment 1

[0033] Example 1. Screening and optimization of oligonucleotide probes for detecting eight SNP sites of CYP450

[0034] 1. Preparation of oligonucleotide gene chips and design and screening optimization of oligonucleotide probes

[0035] The probes are respectively designed on the DNA sequences of each subtype of the human CYP450 enzyme system, and have high sensitivity and specificity. The oligonucleotide probe sequence is consistent with the base sequence of the DNA positive strand in the specific region of the above gene. When the probe is synthesized, its 3' terminal amino group is modified, which is used for the probe to react with the active aldehyde group on the surface of the slide and fix it to the surface of the carrier. After the candidate oligonucleotide probes are purified and quantified, they are spotted on the aldehyde-activated glass slides with a gene chip spotter to make a probe microarray.

[0036] The PCR product using genomic DNA as a template was purifie...

Embodiment 2

[0041] Example 2. Specificity and Sensitivity Evaluation of Oligonucleotide Probes

[0042] 1. Gene chip preparation Dilute the oligonucleotide probes with spotting solution (6×SSC, 0.1% SDS) to a final concentration of 50 μmol / L, and transfer 5ul to a 384-well plate. The probes were spotted on the aldehyde-based chip with a Cartesian chip preparation instrument (see Figure 3 for the arrangement of the probes, and see Figure 5 for the hybridization results of the chips with fluorescently labeled PCR products of CYP450 enzyme genomic DNA).

[0043] Specific detection of hotspot mutation site CYP450 oligonucleotide chip

[0044] Under the optimized conditions of two sets of double PCR amplification and hybridization reaction, 36 clinical DNA samples were detected, and four sets of double PCR amplification were carried out with the DNA samples as templates, and the products were hybridized with the chip. The probes in which signals appear correspond to the fragments of their hyb...

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Abstract

The present invention provides a set of oligonucleotide probe for detecting CYP450 enzyme gene hot mutant site and its uses, belonging to clinical molecular diagnosis field. The probe is designed at whole gene sequence of each subtype enzyme of CYP450 and has relative high sensitivity and specifity. The present invention also provides a gene chip for detecting cytochrome P450 enzyme series mutant sites and can detect gene typing of DNA specimen. The inventive probe can be used in P450 genetype diagnosi, clinical medicament and preventing drug adverse reaction.

Description

technical field [0001] The invention belongs to the technical category of nucleic acid detection of DNA genotype. Specifically, the present invention relates to a set of enzymes capable of detecting subtypes CYP1A2*1F, CYP1A2*1C, CYP2C9*3, CYP2C19*2, CYP2C19*3, CYP2D6*10, CYP2D6*2 in the human liver cytochrome CYP450 enzyme system and CYP3A4*18 representative mutation site genotype oligonucleotide probes, the sequence and use of this set of probes. Background technique [0002] Cytochrome P450 is widely found in animals, eukaryotic organisms, plants, fungi and bacteria and is an essential structural enzyme. Cytochrome P450 is involved in the phase I reaction of drug degradation in the liver. It is estimated that 60% of commonly prescribed drugs require biotransformation through the cytochrome P450 system. The cytochrome P450 superfamily can be divided into families, subfamilies and subtypes in turn. Humans have identified 357 cytochrome P450 genes and 33 pseudogenes, whi...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 王升启文思远许力
Owner INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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