PCR primer design for detection of SNPs in SLC22A1 rs683369 encoding OCT1 as the main transporter of metformin
Abstract
This study aimed to obtain the optimal primer and PCR condition to amplify DNA template of rs683369. DNA was isolated from the human blood specimen. Primer was designed using PrimerBLAST followed by an analysis using OligoAnalyzer, and position flanked by Primer3. The PCR condition was optimized to gain template DNA with good quantity and quality. Sensitivity test of PCR condition was conducted based on the volume and quality of DNA template that has gone through repeated freeze-thaw. Obtained primer pairs were forward: 5’-CCTCCTCTTGCCGTGGTATG-3’ and reverse: 5’-CTGCAAAGTAGCCAACACCG-3’. PCR condition consisted of 1 pre-denaturation cycle (94°C, 2 minutes), 30 consecutive cycles of denaturation (94°C, 40 seconds), annealing (57.4°C, 40 seconds), and elongation (72°C, 1 minute) as well as 1 cycle of post-elongation (72°C, 1 minute). Sensitivity test indicated that the PCR condition could be used for 0.01 μL DNA template volume with 6 cycles of freeze-thaw.
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