Optimization of PCR conditions for amplification of GC-Rich EGFR promoter sequence.
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 MgCl2 concentration ranged from 1.5 to 2.0 mM.


CONCLUSIONS
In conclusion, EGFR promoter region is a difficult PCR target, but it could be amplified after optimization of MgCl2 concentration and annealing temperature in the presence of DMSO and the DNA template of acceptable concentration.
Faculty of Medical Sciences, University of Kragujevac, Kragujevac, Serbia; Faculty of Science, Institute of Biology and Ecology, University of Kragujevac, Kragujevac, Serbia.
Jasmina
J
Obradovic
Vladimir
V
Jurisic
Natasa
N
Tosic
Jasminka
J
Mrdjanovic
Branislav
B
Perin
Sonja
S
Pavlovic
Natasa
N
Djordjevic
D001482:Base Composition / Q000235:genetics*; D004247:DNA / Q000032:analysis / Q000235:genetics; D004121:Dimethyl Sulfoxide; D018773:Genes, erbB-1 / Q000235:genetics*; D060005:Genotyping Techniques / Q000379:methods*; D006801:Humans; D008175:Lung Neoplasms / Q000737:chemistry / Q000235:genetics; D015636:Magnesium Chloride; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D017422:Sequence Analysis, DNA
D015636:Magnesium Chloride; D004247:DNA; D004121:Dimethyl Sulfoxide
EGFR; GC-rich; PCR; genotyping; optimization