Open AccessOptimization of PCR Conditions for Amplification of GC‐Rich EGFR Promoter Sequence
Author(s) -
Obradovic Jasmina,
Jurisic Vladimir,
Tosic Natasa,
Mrdjanovic Jasminka,
Perin Branislav,
Pavlovic Sonja,
Djordjevic Natasa
Publication year - 2013
Publication title -
journal of clinical laboratory analysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 50
eISSN - 1098-2825
pISSN - 0887-8013
DOI - 10.1002/jcla.21632
Subject(s) - microbiology and biotechnology , polymerase chain reaction , primer dimer , agarose gel electrophoresis , gc content , multiple displacement amplification , dna , chemistry , genomic dna , hot start pcr , cytosine , guanine , agarose , biology , gene , multiplex polymerase chain reaction , dna extraction , biochemistry , nucleotide , genome
Background Polymerase chain reaction (PCR) is an extremely sensitive method that often demands optimization, especially when difficult templates need to be amplified. The aim of the present study was to optimize the PCR conditions for amplification of the epidermal growth factor receptor ( EGFR ) promoter sequence featuring an extremely high guanine‐cytosine (GC) content in order to detect single nucleotide polymorphisms ‐216G>T and ‐191C>A. Methods Genomic DNA used for amplification was extracted from formalin‐fixed paraffin‐embedded lung tumor tissue and PCR products were detected by agarose gel electrophoresis. Results Results showed that addition of 5% dimethyl sulfoxide (DMSO), as well as DNA concentration in PCR reaction of at least 2 μg/ml, were necessary for successful amplification. Due to high GC content, optimal annealing temperature was 7°C higher than calculated, while adequate MgCl 2 concentration ranged from 1.5 to 2.0 mM. Conclusion In conclusion, EGFR promoter region is a difficult PCR target, but it could be amplified after optimization of MgCl 2 concentration and annealing temperature in the presence of DMSO and the DNA template of acceptable concentration.