CYP2B6 gene polymorphism detection specific primers and liquid chip
A gene polymorphism and detection solution technology, which is applied in the field of molecular biology, can solve the problems that cannot meet the practical application and cannot be used, and achieve the effects of avoiding uncertain factors, consistent detection results, and simple steps
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Embodiment 1
[0023] Example 1 CYP2B6 gene polymorphism detection liquid chip mainly includes:
[0024] 1. ASPE Primers
[0025] Specific primer sequences were designed for wild-type and mutant types of six common genotypes of CYP2B6 gene, C123T, A101G, G118T, A77G, T139C and C288T. ASPE primers consist of "Tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:
[0026] ASPE primer sequence (Tag sequence+specific primer sequence) of table 1 CYP2B6 gene
[0027]
[0028]
[0029] Each ASPE primer includes two parts, the 5' end is a specific tag sequence for the anti-tag sequence on the corresponding microsphere, and the 3' end is a mutant or wild-type specific primer fragment (as shown in the above table 1). All ASPE primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. Each synthesized primer was prepared into a stock solution of 100 pmol / mL with 10 mmol / L Tris Buffer.
[0030] 2. Microspheres coated with an...
Embodiment 2
[0043] Example 2 Using the CYP2B6 gene polymorphism detection liquid chip described in Example 1 to detect samples The formulations of the various solutions are as follows:
[0044] 50mM MES buffer (pH5.0) formula (250ml):
[0045]
[0046] 2×Tm hybridization buffer
[0047] Reagent
[0048] Store at 4°C after filtration.
[0049] ExoSAP-IT kit was purchased from US USB Company.
[0050] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.
[0051] 1. Sample DNA extraction:
[0052] Refer to the relevant methods of DNA extraction in "Molecular Cloning" to obtain the DNA to be detected.
[0053] 2. PCR amplification of samples to be tested
[0054] Five pairs of primers were designed, and multiplex PCR amplified five target sequences containing six common genotypes C123T, A101G, G118T, A77G, T139C and C288T of CYP2B6 gene in one step. They are 356bp, 235bp, 246bp, 275bp and 384bp respectively, and the primer seque...
Embodiment 3
[0100] Example 3 Detection of CYP2B6 gene polymorphism site by liquid chip with different ASPE primers
[0101] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)
[0102] Taking the CYP2B6 gene A101G and T139C site mutation detection liquid chip as an example, the specific primer sequence of the 3' end of the ASPE primer was designed for the wild type and mutant type of A101G and T139C, and the Tag sequence at the 5' end of the ASPE primer was selected from SEQ ID NO.1-SEQ ID NO.12, correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected from SEQ ID NO.25-SEQ ID NO.36. The specific design is shown in the following table (Table 8). The synthesis of ASPE primers, microspheres coated with anti-tag sequences, amplification primers, detection methods, etc. are as described in Example 1 and Example 2.
[0103] Table 8 Design of liquid phase chip preparation
[0104]...
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