Primer and kit for detecting human low oxygen tolerance related gene polypeptide
A gene polymorphism and kit technology, which is used in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc. And other issues
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Embodiment 1
[0059] Embodiment 1: the preparation of kit (30 tests / box)
[0060] 1. Design and synthesis of primers and probes
[0061]For the polymorphic sites EGLN1 RS479200 and EGLN1RS480902 detected in the human EGLN1 gene, specific mutation sites were selected, and primers were designed using PyroMark Assay Design2.0 software; the forward amplification primer, reverse amplification primer and sequencing primer were first Purified by PAGE and then purified by HPLC, wherein the 5' of the reverse amplification primers of EGLN1 RS479200 and EGLN1 RS480902 are labeled with biotin.
[0062] Table 1. Mutation site and type
[0063] mutation site base change EGLN1 (RS479200) C>T EGLN1 (RS480902) C>T
[0064] The amplified sequence is shown in Table 2:
[0065] Table 2. Specific amplification primers and primer sequences
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[0068] 2. Composition of PCR reaction solution
[0069] Table 3. Composition of PCR reaction solution 1
[0070] ...
Embodiment 2
[0088] Embodiment 2: the use of kit
[0089] 1. Sample testing
[0090] 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 Taq DNA polymerase, aliquot the system, add sample DNA, blank control substance or positive control substance as templates, and form PCR reaction system. Perform PCR amplification according to the PCR reaction procedure.
[0091] The main components of the EGLN1 RS479200 and EGLN1 RS480902 systems are as follows:
[0092] Table 9. Main components of EGLN1 RS479200 system
[0093]
[0094] Table 10. Main components of EGLN1 RS480902 system
[0095]
[0096] The system reaction procedure is as follows:
[0097] Table 11. PCR reaction program
[0098]
[0099]
[0100] After the amplification is completed, check the PCR results on agarose gel to pro...
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