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Optimization of PCR conditions for amplification of GC-Rich EGFR promoter sequence.

ABSTRACT: 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 ?g/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.

SUBMITTER: Obradovic J 

PROVIDER: S-EPMC6807403 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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Optimization of PCR conditions for amplification of GC-Rich EGFR promoter sequence.

Obradovic Jasmina J   Jurisic Vladimir V   Tosic Natasa N   Mrdjanovic Jasminka J   Perin Branislav B   Pavlovic Sonja S   Djordjevic Natasa N  

Journal of clinical laboratory analysis 20131101 6

<h4>Background</h4>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.<h4>Methods</h4>Genomic DNA used for amplificatio  ...[more]

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