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Test chipe of cytochrome P450 gene hereditary variation and its application

A technology of cytochrome and detection chip, applied in the field of detection chip of genetic variation of cytochrome P450 gene, which can solve the problem of lack of functional variation

Inactive Publication Date: 2007-02-14
SHANGHAI BIOCHIP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the other hand, although CYP3A4 metabolizes the most drugs, due to the lack of functional variation and the extensive substrate overlap with CYP3A5, the polymorphic expression of CYP3A5 basically represents the metabolic differences of CYP3A among individuals

Method used

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  • Test chipe of cytochrome P450 gene hereditary variation and its application
  • Test chipe of cytochrome P450 gene hereditary variation and its application
  • Test chipe of cytochrome P450 gene hereditary variation and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1 Preparation of gene chip

[0079] 1. Probe Dissolution

[0080] Each probe was diluted with TE solution to a final concentration of 10 mM. Mix the probe with a concentration of 10mM and the PBS solution with a concentration of 200mM in a medium volume of a 384-well plate, seal the 384-well plate with an adhesive sheet, shake at room temperature for 2 minutes, centrifuge, and store at -20°C for sample application .

[0081] 2. Spotting

[0082] The pre-designed and synthesized probes are loaded onto the solid-phase carrier substrates made of glass slides, silicon wafers, etc. through contact spotting or inkjet spotting. The film base adopts Cell Associates CSS-100 aldehyde base film base, and the Ominigrid 100 model spotting instrument of GeneMachine Company is applied at a humidity of 65-75% (based on the FullMoon film base) and a temperature of 25°C. The format of spotting is 1×3, and each matrix is ​​8×18. After the spotting is completed, leave the chip ...

Embodiment 2

[0083] Embodiment 2 Processing and labeling of samples to be tested

[0084] 1. Human genomic DNA extraction and target gene amplification

[0085] Using the FlexiGene DNA Kit (250) (QIAGEN, Cat.No.51206) kit to extract genomic DNA from human peripheral blood, the specific steps are as follows:

[0086] (1) Mix ACD (citric acid 8g / L, sodium citrate 22g / L, glucose 24.5g / L) anticoagulated peripheral whole blood, take 1ml into a 15ml centrifuge tube, add 2.5ml buffer FG1, fully Mix upside down;

[0087] (2) Centrifuge at 2000 g for 5 minutes, discard the supernatant. Let stand for 2 minutes;

[0088] (3) Add 500ul buffer FG2 / QIAGEN protease mixed reagent, mix immediately until the original precipitate completely disappears;

[0089] (4) Water bath at 65°C for 10 minutes, shake at a constant speed at 40 times / min;

[0090] (5) Add 500ul 100% isopropanol and mix thoroughly until visible flocculent DNA precipitates;

[0091] (6) Centrifuge at 3000g for 5 minutes, discard the s...

Embodiment 3

[0114] Example 3 Hybridization, washing and result detection

[0115] Fluorescence-labeled PCR products were denatured at 95°C for 10 minutes, and immediately placed on ice for hybridization. The hybridization reaction system included:

[0116]

[0117] The reaction conditions were incubation at 48°C for 120 min, followed by 1× wash buffer I (5×SSC, 0.1% SDS), 1× wash buffer II (2×SSC, 0.1% SDS) and 1× wash buffer III ( 1×SSC) at 42°C for 10 min each, and finally with ddH2O for 0.5 min.

[0118] After washing, the chips were dried and scanned with a GenePix 4000B confocal laser scanner (other laser scanners can also be used). The hybridization results obtained by scanning the hybridized chips are as follows: Figure 4 As shown, then use GenePix Pro to process the image to obtain the data file, and then analyze the data file to obtain the genotype of the target gene. Figure 4 The typing results shown are CYP2D6*10, CYP2C19*1, CYP2C9*1 and CYP3A5*3.

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Abstract

The invention discloses a cytochrome P450 gene genetic variation detecting chip. It includes the probe fixed on the solid phase carrier to hybridize with the cytochrome P450 gene nucleotide sequence and / or complementation sequence. The invention also discloses the used chip detecting method. The detecting chip can effectively detect subtype genetic variation of CYP2C9, CYP2C19, CYP2D6, CYP3A5, forecast the therapeutic effectiveness of over 40% clinic common medicine to realize individualization medical treatment.

Description

technical field [0001] The invention relates to a drug gene detection chip, in particular to a detection chip of cytochrome P450 gene genetic variation related to human drug metabolism and its application. Background technique [0002] For a long time, clinicians often take the same drug at the same dose for the same disease based on the clinical trials of the drug, or make slight adjustments to the dose based on clinical experience. In the clinical treatment of diseases, it often happens that the same drug produces very different curative effects when treating different patients with the same disease, and some patients even have serious side effects or no response, which not only causes huge waste, but sometimes directly delays the disease. Timely treatment, delayed disease. The annual death rate and corresponding health resource expenditure caused by adverse drug reactions and drug-induced diseases are extremely staggering, and the annual cost in this area in the United S...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 盛海辉肖华胜
Owner SHANGHAI BIOCHIP
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