Effects of different additives for detection of single nucleotide polymorphisms in promoter sequence EGFR gene in NSCLC.

Abstract

Background: The aim of this study was to test the effects of several additives in various concentrations for amplification single nucleotide polymorphisms in promoter sequence of the epidermal growth factor receptor (EGFR), since the promoter sequence containing the multiple GC regions which are difficult for the amplification. Material and Methods: PureLink™ Genomic DNA Kits (Invitrogen/ LifeTechnologies, Carlsbad, CA) were used for extracion of DNA from formalinfixedparaffin- embedded lung cancer tissue. EGFR polymorphisms −216G>T and −191C>A were genotyped using the PCR-RFLP method. Sequencing was conductedusing ABI PRISM® BigDyeTM Terminator v 3.1 Cycle Sequencing Kit in both forwardand reverse direction. Results: Between several tested additive including; glycerol, DMSO, formamide, Tween 20, Triton X-100, PEG and BSA, only a two show effectiveness including glycerol and DMSO, and with best results at concentrations of 15% and 5% respectively. Comparison of the obtained sequence with the reference sequence of EGFR promoter region (http://www.ncbi.nlm.nih.gov; GenBank reference: M11234.1) revealed that the PCR amplification was highly specific. Conclusions: In this study we have shown that using appropriate cosolvent it ispossible amplify promotor region of EGFR for single nucleotide polymorphisms −216G>T or −191C>A.