DNA micro-array chip, detection method thereof and uses in CYP3A4, CYP3A5 and MDR1 gene polymorphism detection

A microarray chip, gene technology, applied to the application of CYP3A5 and MDR1 gene polymorphisms, DNA microarray chip and its detection, detection of CYP3A4 field, can solve the problem of low detection throughput, cost and time consuming, practical for clinical detection and diagnosis It is easy to prepare, easy to operate, and realize the effect of personalized medicine.

Inactive Publication Date: 2009-07-15
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] At present, domestic and foreign studies on CYP3A4, CYP3A5 and MDR1 gene polymorphisms are basically aimed at the detection of a single SNP site of a single gene, and cannot perform comprehensive analysis on the detection of multiple sites at the same time, so multiple tests are often required to obtain The detection process of these genetic polymorphisms is complicated and time-consuming
Due to the limited genotypes that can be analyzed, the practicability of clinical detection and diagnosis is low; and due to the low detection throughput, it is difficult to apply to personalized medicine
So far, there are no reports on multi-sample one-time detection of the three indicators of CYP3A4, CYP3A5 and MDR1 gene polymorphisms

Method used

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  • DNA micro-array chip, detection method thereof and uses in CYP3A4, CYP3A5 and MDR1 gene polymorphism detection
  • DNA micro-array chip, detection method thereof and uses in CYP3A4, CYP3A5 and MDR1 gene polymorphism detection
  • DNA micro-array chip, detection method thereof and uses in CYP3A4, CYP3A5 and MDR1 gene polymorphism detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 DNA microarray chip probe and PCR primer design

[0045] 1. CYP3A4 (Cytochrome P4503A4)

[0046] Design 20 kinds of CYP3A4 gene polymorphism detection probes, covering 10 gene polymorphism sites, and their nucleotide sequences are shown in SEQ ID NO:1~SEQ ID NO:20; PCR amplification of CYP3A4 gene in the target sample In addition, 7 upstream primers are designed, and the upstream primers are fluorescently labeled with CY3, and 7 downstream primers are designed, totaling 14 primers. The nucleotide sequences are shown in SEQ ID NO:50~SEQ ID NO:63.

[0047] 2. CYP3A5 (Cytochrome P4503A5)

[0048] Design CYP3A5 design 20 kinds of gene polymorphism detection probes, covering 10 gene polymorphism sites, the nucleotide sequence of which is shown in SEQ ID NO: 21 ~ SEQ ID NO: 40; PCR of CYP3A5 gene in the target sample For the amplification, 7 upstream primers are designed, and the upstream primers are fluorescently labeled with CY3, and 7 downstream primers are designed,...

Embodiment 2

[0051] Example 2 Preparation of DNA microarray chip

[0052] Spot probe solution: Dilute the probe with 5×SSC (containing 0.05% by mass SDS) solution until the final concentration of the probe is 30 μM; SSC buffer is commonly used by those in the art, and its preparation method is common in the art Known.

[0053] Spotting matrix: Using a gene chip automatic spotting instrument, 49 detection probes and 1 control probe designed in Example 1 (used for internal control of the chip surface chemical modification, spotting and immobilization process), Manufactured to a specific area of ​​the solid carrier (glass slide), such as figure 1 The prepared DNA microarray chip shown has 10 microarray matrices on the solid carrier.

[0054] figure 2 Shown is the arrangement of the probes in each microarray matrix. The above 49 probes are represented by their nucleotide sequence numbers SEQ ID NO: 1 ~ SEQ ID NO: 49, and the matrix also contains 1 control Probe (FC). From figure 2 It can be se...

Embodiment 3

[0056] Example 3 Detection of clinical samples by DNA microarray chip

[0057] Take 8 samples, extract the genomic DNA of the samples using conventional methods in the field, and perform DNA sequencing to determine the results of the CYP3A4, CYP3A5 and MDR1 gene polymorphism mutations in the genome, and then use the DNA microarray chip prepared in Example 2 Perform testing and verification.

[0058] The DNA microarray chip prepared in Example 2 was used for one-time detection of the above 8 samples.

[0059] A total of 36 primers of CYP3A4, CYP3A5 and MDR1 genes in the target sample designed in Example 1 were synthesized, dissolved and mixed, and divided into 10 tubes, of which 8 tubes were respectively added with the genomic DNA of the above 8 samples, 1 tube As a DNA dissolving solution (negative control), 1 tube is added with a DNA of known CYP3A4, CYP3A5 and MDR1 genotypes (positive control), and the rest of the PCR reaction components in 10 tubes are in accordance with the PC...

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Abstract

The invention discloses a DNA micro-array chip and a detection method thereof and the application on detecting the polymorphism of CYP3A4, CYP3A5 and MDR1 genes. The chip comprises a solid phase carrier and a probe. The nucleotide sequence of the probe is shown as SEQ ID No: 1-SEQ ID No: 49. The probe is firstly arranged on the solid phase carrier. And then the target nucleotide sequence of a sample to be detected is marked and prepared. After that, the target nucleotide sequence is hybridized with the probe on the chip. Finally, the hybridization result is detected to obtain the information of the CYP3A4, CYP3A5 and MDR1 genes in the sample to be detected. The chip of the invention can be used for the detection of a plurality of genes of a plurality of samples simultaneously once only and has the advantages of simple operation and high result accuracy. Large amount of genetic information of patients can be obtained through detection once. The DNA micro-array chip and the detection method thereof can be used for instructing the formulation of reasonable drug schemes and realizing the personalized medical treatment.

Description

Technical field [0001] The invention relates to medical in vitro diagnostic technology, in particular to a DNA microarray chip and its detection method and its application in detecting CYP3A4, CYP3A5 and MDR1 gene polymorphism. Background technique [0002] Organ transplantation is the most effective method for the treatment of end-stage liver disease. The wide application of commonly used clinical immunosuppressants, tacrolimus, cyclosporine and rapamycin, has improved the clinical effect of organ transplantation, and its side effects have also attracted increasing attention. [0003] In clinical applications, it is likely that insufficient dosing may result in "insufficient immunosuppression" resulting in graft rejection and result in transplant failure, or excessive dosing may result in "over-immune suppression" leading to liver and kidney toxicity, infection and tumors. At present, the traditional "one drug per thousand people, one dose per thousand people" administration mod...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 陈规划姜楠杨扬李华黄民王雪丁林炳生张帆
Owner SUN YAT SEN UNIV
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