Desain Primer Untuk Deteksi SNP RS683369 SLC22A1 Penyandi Organic Cation Transporter 1 Sebagai Transporter Utama Metformin Menggunakan Polymerase Chain Reaction

Abstract

Main therapy for Diabetic Melitus Type 2 (DM2) is usinf Metformin which is a strong base in body fisiologis pH and in cationic form >99%, so it needs a transporter to penetrate cell membran. Main transporter to distribute metformin to hepatocyte is Organic Cation Transporter 1 (OCT1). Polymorphism SNP rs683369 SLC22A1 in OCT1 known has a clinical effect in pharmacodynamic and pharmacokinetic profile of metformin. Hence, design an appropriate primer become an important part in genomic polymorphism analysis to give consideration therapy in management DM2. The aim of this study was to find primers, an optimal PCR condition to support analysis rs683369 and sensitivity level of an optimal PCR condition in supporting analysis rs683369 to the variation of volume and quality of template DNA based on the freeze-thawing frequency. DNA was isolated from DM2 patients. Primer was designed using website Primer-BLAST and aalyzed using website primer3 dan oligoanalyser to get the most spesific primer. Condition for DNA amplification using PCR was optimized to obtain the best quality and amount of DNA template. Then, the sensitivity level of amplification condition using PCR was obtained towards the difference volume and quality of DNA template that has been frozen and thawed many times based on electrophoresis visualization. Primer pair (forward : 5’–CCT CCT CTT GCC GTG GTA TG-3’ dan reverse : 5’- CTG CAA AGT AGC CAA CAC CG-3') which is the result of this study showed that it can use to amplify 128bp of SNP rs683369 SLC22A1. It amplifies for 1 cycle of pre-denaturation at 94oC for 2 minutes, 30 cycles of denaturation at 94C for 40 seconds, annealing at 57.4C for 40 seconds and extention at 72C for 1 minutes; and 1 cycle post-extention at 72oC for 1 minute. This condition could amplify up to 0,01μL of DNA template which has been frozen and thawed for 6 cycles.