CYP2D6 gene mutation detection liquid-phase chip and detection method
A technology of CYP2D6C100T and CYP2D6G1846A, applied in CYP2D6 gene mutation detection liquid chip, medical and biological fields, can solve the problems of poor repeatability of test results, high price, low sensitivity, etc., achieve accurate and reliable test results, improve detection sensitivity, The effect of high specificity and accuracy
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Embodiment 1
[0030] Example 1 CYP2D6 gene mutation detection liquid chip mainly includes:
[0031] 1. ASPE Primers
[0032] The two normal genotypes of CYP2D6 gene are CYP2D6*1 and CYP2D6*2, and CYP2D6*2 is C2850T mutation. The five domestic common mutation genotypes are CYP2D6*3, CYP2D6*4, CYP2D6*5, CYP2D6*10 and CYP2D6 gene duplication mutation. CYP2D6*3 is the A2549del deletion mutation, CYP2D6*4 is the common mutation of C100T and G1846A, CYP2D6*5 is the entire gene deletion mutation, CYP2D6*10 is the C100T mutation, and there are also duplication mutations of the entire gene. Specific primers were designed for the two normal genotypes CYP2D6*1 and CYP2D6*2 of the CYP2D6 gene and the gene duplication mutations of the five domestic common mutant genotypes CYP2D6*3, CYP2D6*4, CYP2D6*5, CYP2D6*10 and CYP2D6 sequence. ASPE primers consist of "Tag + specific primer sequence". ASPE primer sequences are shown in the table below:
[0033] Table 1 ASPE primer sequence (Tag+ specific primer...
Embodiment 2
[0050] Example 2 Detection of Clinical Samples Using CYP2D6 Gene Mutation Detection Liquid Chip
[0051] The formula of described various solutions is as follows:
[0052] 50mM MES buffer (pH5.0) formula (250ml):
[0053] Reagent
[0054] 2×Tm hybridization buffer
[0055] Reagent
[0056] Store at 4°C after filtration.
[0057] ExoSAP-IT kit was purchased from US USB Company.
[0058] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.
[0059] 1. Sample DNA extraction:
[0060] Refer to the instructions of the AxyPrep Whole Blood Genome Mini Extraction Kit to obtain the DNA to be detected.
[0061] 2. PCR amplification of samples to be tested
[0062]Use Primer5.0 to design six pairs of primers, divide them into two tubes for PCR reaction, one tube is nested PCR, use SEQ NO.31-32 to amplify the fragments containing C100T, G1846A, A2549del and C2850T sites, and then use SEQ NO. NO.33-34 amplifies the fr...
Embodiment 3
[0128] Example 3 Detection of CYP2D6 C2850T, CYP2D6 Deletion Gene Mutation Detection Liquid Chip on Clinical Samples
[0129] Use CYP2D6 C2850T, CYP2D6 Deletion gene mutation detection liquid chip to detect serum samples 1-20, the synthesis of ASPE primers, Anti-tag sequence coated microspheres, amplification primers, detection methods, etc. are as described in Example 1 and Example 2 mentioned.
[0130] Table 6 Sample test results (MFI)
[0131] serial number
[0132] serial number
[0133] Table 7 Analysis results of CYP2D6 C2850T and CYP2D6 Deletion gene mutations in samples
[0134] serial number
[0135] serial number
[0136] It can be seen from the results of Example 2 and Example 3 that only CYP2D6 C2850T and CYP2D6 Deletion gene mutations in Example 3 are detected by the liquid chip and the results are consistent with those in Example 2.
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