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. 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.
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. 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.
Authors: Charles M Rudin; Wanqing Liu; Apurva Desai; Theodore Karrison; Xuemin Jiang; Linda Janisch; Soma Das; Jacqueline Ramirez; Balasubramanian Poonkuzhali; Erin Schuetz; Donna Lee Fackenthal; Peixian Chen; Deborah K Armstrong; Julie R Brahmer; Gini F Fleming; Everett E Vokes; Michael A Carducci; Mark J Ratain Journal: J Clin Oncol Date: 2008-03-01 Impact factor: 44.544