Optimization of the condition for PCR-directed sequencing of microsatellites poly Adenine (A) length polymorphisms is more difficult and sensitive compared with other common sequences. Replication slippage may occur for polymerase enzyme during microsatellite amplification and direct sequencing of these PCR products will be challenging for heterozygote samples. So, the aim of this study is to introduce optimal condition for amplification of microsatellites poly (A) length polymorphisms such as variation of poly(A) at the 3′-end of the vitamin D receptor (VDR) gene. In this study firstly, we analyzed various types of microsatellite sequences by in silico method to select a short microsatellite sequence which has various alleles with a little length difference. Finally, VDR poly(A) microsatellite was selected. Amplification of this locus was optimized by applying hot-start PCR using gradients of temperature, DMSO, MgCl2, Taq polymerase, and primers concentration. Crush and soak method was used for DNA extraction of alleles in heterozygote samples from polyacrylamide gel. PCR products were analyzed by using single-strand conformation polymorphism and PCR-direct sequencing. Our observations showed that hot-start PCR was optimized in the concentration of 0.2 mM of primers, 0.8mM of MgCl2 and 1.75 unit of Taq polymerase in 25 μl PCR mixtures. These optimal conditions lead to remove Taq polymerase slippage and non-specific PCR product. Considering the possible errors in amplification and analysis of microsatellite, the presented method in this study could be an efficient strategy in analysis of these sequences.
