Specific primer and liquid chip for detecting polymorphism of DPYD (dihydropyrimidine dehydrogenase) gene
A gene polymorphism and detection solution technology, applied in the field of molecular biology, can solve the problems of unsatisfactory practical application, poor timeliness, low sensitivity, etc., and achieve the effect of improving detection accuracy, consistent detection effect, and simple steps
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Embodiment 1
[0029] Example 1 DPYD gene polymorphism detection liquid chip mainly includes:
[0030] 1. ASPE Primers
[0031] Specific primer sequences were designed for wild-type and mutant types of three common genotypes of DPYD gene, G55A, G141A and G134T. ASPE primers consist of "tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:
[0032] Table 1 ASPE primer sequence of DPYD gene (tag sequence + specific primer sequence)
[0033]
[0034] 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.
[0035] 2. Microspheres coated with anti-tag sequences
[0036]According...
Embodiment 2
[0048] Example 2 Detection of samples using the DPYD gene polymorphism detection liquid chip described in Example 1
[0049] The formula of described various solutions is as follows:
[0050] 50mM MES buffer (pH5.0) formula (250ml):
[0051]
[0052] 2×Tm hybridization buffer
[0053] Reagent
[0054] Store at 4°C after filtration.
[0055] ExoSAP-IT kit was purchased from US USB Company.
[0056] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.
[0057] 1. Sample DNA extraction:
[0058] Refer to the relevant methods of DNA extraction in "Molecular Cloning" to obtain the DNA to be detected.
[0059] 2. PCR amplification of samples to be tested
[0060] Three pairs of primers were designed, and multiplex PCR amplified three target sequences containing three common genotypes G55A, G141A, and G134T of the DPYD gene in one step. The product sizes were 231bp, 192bp, and 134bp. ) are shown in Table 3 above.
[0...
Embodiment 3
[0102] The liquid phase chip of embodiment 3 different ASPE primers is to the detection of DPYD gene SNP site
[0103] 1. Design of liquid phase chip preparation (selection of tag sequence and Anti-tag sequence)
[0104] Taking the liquid phase chip for detecting mutations at the G55A site of the DPYD gene as an example, the specific primer sequences at the 3' end of the ASPE primer were designed for the wild type and mutant type of G55A, and the tag sequence at the 5' end of the ASPE primer was selected from SEQ ID NO.1 - SEQ ID NO.6. Correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected from SEQ ID NO.13-SEQ ID NO.18. The specific design is shown in the following table (Table 7). 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.
[0105] Table 7 Design of liquid phase chip preparatio...
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