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A dna microarray chip and its detection method and its application in the detection of cyp3a4, cyp3a5 and mdr1 gene polymorphisms

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

Inactive Publication Date: 2011-12-28
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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  • A dna microarray chip and its detection method and its application in the detection of cyp3a4, cyp3a5 and mdr1 gene polymorphisms
  • A dna microarray chip and its detection method and its application in the detection of cyp3a4, cyp3a5 and mdr1 gene polymorphisms
  • A dna microarray chip and its detection method and its application in the detection of cyp3a4, cyp3a5 and mdr1 gene polymorphisms

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Design of DNA Microarray Chip Probes and PCR Primers

[0045] 1. CYP3A4 (cytochrome P4503A4)

[0046] Design 20 kinds of CYP3A4 gene polymorphism detection probes, covering 10 gene polymorphism sites, the nucleotide sequences of which are shown in SEQ ID NO: 1~SEQ ID NO: 20; the PCR amplification of CYP3A4 gene in the target sample In addition, 7 upstream primers were designed, and the upstream primers were labeled with CY3 fluorescence, and 7 downstream primers were designed, a total of 14 primers, the nucleotide sequences of which 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 sequences of which are shown in SEQ ID NO: 21 ~ SEQ ID NO: 40; the PCR of CYP3A5 gene in the target sample For amplification, 7 upstream primers were designed, and the upstream primers were labeled with CY3 fluorescence, ...

Embodiment 2

[0051] Example 2 Preparation of DNA Microarray Chip

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

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

[0054] figure 2 Shown is the arrangement of probes in each microarray matrix, wherein the above 49 probes are represented by their nucleotide sequence numbers SEQ ID NO: 1 to SEQ ID NO: 49, and the matrix also contains ...

Embodiment 3

[0056] Example 3 DNA microarray chip detection of clinical samples

[0057] Take 8 samples, use the conventional methods in the field to extract the genomic DNA of the samples, and perform DNA sequencing to determine the polymorphic mutation results of CYP3A4, CYP3A5 and MDR1 genes in the genome, and then use the DNA microarray chip prepared in Example 2 to compare Perform detection verification.

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

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

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Abstract

The invention discloses a DNA microarray chip, its detection method and its application in detecting CYP3A4, CYP3A5 and MDR1 gene polymorphisms. The chip includes a solid phase carrier and a probe, and the nucleotide sequence of the probe is as the probe The nucleotide sequences are shown in SEQ ID NO: 1 to SEQ ID NO: 49. The present invention first loads the probe on the solid phase carrier, then labels and prepares the target nucleotide sequence of the sample to be tested, then hybridizes the target nucleotide sequence with the probe on the chip, and finally analyzes the hybridization result The detection can obtain the information of CYP3A4, CYP3A5 and MDR1 genes in the sample to be tested. The chip of the present invention can simultaneously detect multiple genes on multiple samples at one time, with simple operation and high accuracy of results, and a large amount of genetic information of patients can be obtained through one detection, which can be used to guide the formulation of rational drug regimens and realize individualization medical.

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 polymorphisms. Background technique [0002] Organ transplantation is the most effective method for the treatment of end-stage liver disease. The widespread use of clinical immunosuppressants tacrolimus, cyclosporine and rapamycin has improved the clinical effect of organ transplantation. At the same time, its toxic and side effects have also attracted increasing attention. [0003] In clinical application, it is very likely that the insufficient dosage of the drug will lead to "insufficient immunosuppression", which will lead to the immune rejection of the graft and lead to graft failure, or the "excessive immunosuppression" caused by excessive drug administration will lead to the occurrence of liver and kidney toxicity, infection and tumor. At present, the tradit...

Claims

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Application Information

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Patent Type & Authority Patents(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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