Peomorphic primer of DPYD*13 gene and detection method thereof
A technology for gene polymorphism and detection method, applied in the field of primers and detection of DPYD*13 gene polymorphism, can solve problems such as unfavorable promotion and application, complicated operation, etc. The effect of the clearance rate
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Embodiment 1
[0023] Embodiment one, primer
[0024] The DPYD*13 gene primers provided by the present invention are shown in Table 1, including PCR amplification primers and SNaPshotPCR primers, and the PCR amplification primers and SNaPshotPCR primers are corresponding. All primer sequences provided by the present invention are compared through UCSC database, and there is no known SNP site.
[0025] Table 1 Primers provided by the present invention
[0026]
Embodiment 2
[0027] Embodiment two, the specificity of primer
[0028] The primers provided by the present invention were blasted in UCSC, and the results were as follows:
[0029] 1) DPYD*13 gene-specific primers were used for Blast in UCSC. The amplified fragments of all primers covered the corresponding detection sites without other homologous genes. The amplified fragment of DPYD*13 gene was located at chr1:97981281-97981471, the length It is 191bp, the amplified sequence diagram is as follows figure 1 .
[0030] 2) Use the SNaPshotPCR primers in Table 1, SNaPshot method for detection, the results are as follows figure 2 As shown, the relative position of each product peak and the bases incorporated in the sequencing reaction were in line with expectations.
Embodiment 3
[0031] Example 3, Detection of DPYD*13 Gene Polymorphism
[0032] 1) Extract DNA samples from EDTA anticoagulated peripheral blood. The extraction method refers to the instructions of TIANampBloodDNAKit (purchased from Tiagen, product number DP318), and the DNA samples are diluted to 100ng / μL for later use.
[0033] 2) Prepare primers for PCR amplification, vortex and mix well; after short centrifugation, use Q5 for PCR amplification ? Hot start ultra-fidelity 2XMasterMix (purchased from NEB Company, product number M0494L), the reaction system is shown in Table 2, shake and mix well, after a short centrifugation, aliquot 18.0 μL into the labeled PCR reaction tube; pour the labeled PCR reaction tube Add 5.0 μL of primer mixture to the tube, and carry out PCR amplification according to the following procedures: stage 1: 98°C for 3 min; stage 2: 98°C for 10 s; stage 3: 58°C for 30 s; stage 4: 72°C for 1 min; stage 5: return to stage 2 , 2: 9 cycles; stage 6: 5 min at 72°C; stage...
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