Sequencing primer for qualitative detection of genetic typing of uridinediphosphoglucuronate glucuronosyltransferase 1A1 and kit thereof
A uridine diphosphate glucose, qualitative detection technology, applied in the direction of recombinant DNA technology, DNA / RNA fragments, etc., to achieve high sensitivity, simple sample processing, and intuitive results
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Embodiment 1
[0043] Embodiment 1: the preparation of kit
[0044] 1. Design and synthesis of primers
[0045] Studies have shown that the current UGT1A1 gene polymorphisms are mainly UGT1A1*6 and UGT1A1*28 gene polymorphisms, UGT1A1*6 gene mutations mainly occur in the 211th nucleotide of the first exon G mutation to A, The polymorphism of UGT1A1*28, which causes the encoded glycine to be changed to arginine (G71R), is the (TA) n repeat sequence promoter, using PyroMark Assay Design2.0 software, according to these two mutation sites (Table 1) The PCR amplification primers and pyrosequencing primers were designed and determined (Table 2). The most important factor affecting the accuracy of the kit is the primers, including amplification primers and sequencing primers. In the early stage of design, we designed Multiple sets of primers were compared (see Figure 9 ); the amplification primers and sequencing primers were purified by PAGE first, and then purified by HPLC, among which UGT1A1*6...
Embodiment 2
[0058] Embodiment 2: the use of kit
[0059] 1. sample amplification
[0060] Take the blank control, positive control and sample DNA respectively as PCR reaction templates, add UNG enzyme, Taq polymerase, specific PCR reverse amplification primers, and PCR reaction solution containing PCR forward amplification primers to form a PCR reaction solution. reaction system for PCR amplification.
[0061] The main components of the UGT1A1*6 system are as follows:
[0062]
[0063] The main components of the UGT1A1*28 system are as follows:
[0064]
[0065] 2. PCR reaction program
[0066] Set up the PCR reaction program, put the reaction tube into the fluorescent PCR instrument (ABI7500) to start amplification, the reaction program is as follows:
[0067] Table 3. PCR reaction program
[0068]
[0069]
[0070] The annealing temperature of detection site 1 (UGT1A1*6) was set at 61°C, and the annealing temperature of detection site 2 (UGT1A1*28) was set at 59°C.
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