Background
Polymerase chain reaction (PCR) is an extremely sensitive method that often demands optimization, especially when difficult templates need to be amplified. The
Conclusion
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.
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.