Primer and method for detecting CYP2D6 gene polymorphism
A gene polymorphism and amplification primer technology is applied in the field of primers for detecting CYP2D6 gene polymorphism, achieving the effects of good specificity, improved therapeutic effect and reduced adverse reactions
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[0025] Embodiment 1, primer
[0026]The primers provided by the present invention are shown in Table 1, including nested PCR amplification primers and SNaPshot PCR primers; the nested PCR amplification primers include nested A round PCR amplification primers and nested B round PCR amplification primers. The nested B-round PCR amplification primers and SNaPshot PCR primers are corresponding. All primer sequences provided by the present invention are aligned through the UCSC database, and there is no known SNP site.
[0027] Table 1 Primers provided by the present invention
[0028]
Example Embodiment
[0029] Embodiment 2. The specificity of primers
[0030] The primers provided by the invention were subjected to Blasting in UCSC, and the results were as follows:
[0031] 1) CYP2D6 gene-specific primers were used for Blast in UCSC without other homologous genes. 2) The amplified fragment of CYP2D6_C2850T is located at chr22:42127836+42128150, the length is 315bp, and the amplified sequence is as follows figure 1 The amplified fragment of CYP2D6_G3183A is located at chr22:42127471-42127709, the length is 239bp, and the amplified sequence is as follows figure 2 ; CYP2D6_G4180C amplified fragment is located at chr22:42126391-42126685, the length is 295bp, and the amplified sequence is as follows image 3 ;CYP2D6_C100T amplified fragment is located at chr22:42130611-42130821, the length is 211bp, and the amplified sequence is as follows Figure 4 ;CYP2D6_G1846A / CYP2D6_G1661C amplified fragment is located at chr22:42128876+42129258, the length is 383bp, the amplified sequenc...
Example Embodiment
[0033] Embodiment 3. Detection of CYP2D6 gene polymorphism
[0034] 1) Extract DNA samples from EDTA anticoagulated peripheral blood. The extraction method refers to the instructions of TIANampBloodDNAKit (purchased from Tiagen, Cat. No. DP318), and the DNA samples are diluted to 100ng / μL for later use.
[0035] 2) Prepare primer mixture: prepare nested A round PCR primer mixture, CYP2D6-Fwd:CYP2D6-Rev:H2O=1:1:3, the final primer concentration is 1pmol / μL, shake and mix well, centrifuge briefly for later use; Preparation B 轮PCR引物混合物,将5对引物如下,CYP2D6_C2850T-Fwd / CYP2D6_C2850T-Rev,CYP2D6_C100T-Fwd / CYP2D6_C100T-Rev,CYP2D6(1661 / 1846)-Fwd / CYP2D6(1661 / 1846)-Rev,CYP2D6_G3183A-Fwd / CYP2D6_G3183A -Rev, CYP2D6_G4180C-Fwd / CYP2D6_G4180C-Rev were mixed in equal volume, and the final primer concentration was 0.5pmol / μL; briefly centrifuged for use; PCR amplification was performed using Q5 ? Hot-start ultra-fidelity 2XMasterMix (purchased from NEB Company, item number M0494L), and the reaction s...
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