Primer pair and kit for detecting CYP3A4 genotyping by pyrosequencing
A technology of pyrosequencing and genotyping, which is applied in the field of primer pairs and kits for detecting CYP3A4 genotyping by pyrosequencing, which can solve the problems of difficulty in meeting clinical testing requirements, low accuracy of testing results, and long testing cycle To achieve real-time monitoring of the reaction process, high-throughput sample detection, and short reaction time
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Embodiment 1
[0040] Embodiment 1: the preparation of kit
[0041] 1. Design and synthesis of primers and probes
[0042] For the polymorphic site CYP3A4*1G detected in the human CYP3A4 gene, select specific mutation sites and use PyroMarkAssayDesign2.0 software to design primers; the forward amplification primers, reverse amplification primers and sequencing primers are first purified by PAGE , and then purified by HPLC, wherein the 5' of SEQ ID NO.1 is biotin-labeled.
[0043] Table 1. Mutation site and type:
[0044] Mutation
base change
CYP3A4*1G (rs2242480)
C>T
[0045] The amplified sequence is shown in Table 2:
[0046] Table 2. Specific amplification primers and primer sequences
[0047]
[0048]
[0049] 2. Selection of reference substance
[0050] A synthetic oligonucleotide chain TAYGGTTTGCAcontrololigo was used as the quality control substance; DNase / RNase-Free water was used as the blank control substance.
[0051] 3. Composition of PC...
Embodiment 2
[0054] Embodiment 2: the use of kit
[0055] 1. Sample testing
[0056] Dissolve the dry powder of the primers (the validity period of the primers is 1 month after dissolution). Prepare the system according to the number of templates: take the PCR reaction solution, add dissolved primers, uracil DNA glycosylase, and TaqDNA polymerase, aliquot the system, add sample DNA, blank control substance or positive control substance as templates, and form a PCR reaction system. Perform PCR amplification according to the PCR reaction procedure.
[0057] The main components of the CYP3A4*1G system are as follows:
[0058] Table 4. Main components of CYP3A4*1G system
[0059]
[0060]
[0061] The system reaction procedure is as follows:
[0062] Table 5. PCR reaction program
[0063]
[0064] After the amplification is completed, check the PCR results on agarose gel to proceed to the next step.
[0065] 2. Pyrosequencing
[0066] Sequencing was performed in accordance wit...
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