Effects of DMSO, glycerol, betaine and their combinations in detecting single nucleotide polymorphisms of epidermal growth factor receptor (EGFR) gene promoter sequence in non-small-cell lung cancer (NSCLC) patients.
The aim of the study was to examine the effects of frequently used polymerase chain reaction (PCR) additives DMSO, glycerol and betaine on amplification of GC-rich epidermal growth factor receptor (EGFR) gene promoter region, in order to detect the presence of -216G>T and -191C>A gene variations in non-small-cell lung cancer (NSCLC) patients. PCR products and restriction fragments were detected by electrophoresis on 8% polyacrylamide gel and 3% agarose gel. Our analysis shows that single used additives including DMSO in concentration of 7% and 10%, glycerol in concentration of 10%, 15% and 20%, as well as betaine in concentration of 1M, 1.5M and 2M significantly enhanced the yield and specificity of PCR reaction. In addition, the combination of 10% DMSO with 15% glycerol has shown positive effects, whereas other analyzed combinations of additives failed to amplify the EGFR promoter region.
Faculty of Medical Sciences, University of Kragujevac, Svetozara Markovica 69, Kragujevac, Serbia. Electronic address: vdvd@lycos.com.
Vladimir
V
Jurišić
Institute of Biology and Ecology, Faculty of Science, University of Kragujevac, Radoja Domanovica 12, Kragujevac, Serbia.
Jasmina
J
Obradović
Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, Belgrade, Serbia.
Natasa
N
Tošić
Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, Belgrade, Serbia.
Sonja
S
Pavlović
University of East Sarajevo, Faculty of Medicine, Foca, Republic of Srpska, Bosnia and Herzegovina.
Milan
M
Kulić
Faculty of Medical Sciences, University of Kragujevac, Svetozara Markovica 69, Kragujevac, Serbia.
Nataša
N
Djordjević
D000328:Adult; D000368:Aged; D001622:Betaine / Q000737:chemistry*; D002289:Carcinoma, Non-Small-Cell Lung / Q000737:chemistry / Q000235:genetics*; D004273:DNA, Neoplasm / Q000737:chemistry / Q000235:genetics; D004121:Dimethyl Sulfoxide / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D066246:ErbB Receptors / Q000737:chemistry / Q000235:genetics*; D005260:Female; D005990:Glycerol / Q000737:chemistry*; D006801:Humans; D008175:Lung Neoplasms / Q000737:chemistry / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D011401:Promoter Regions, Genetic
D004273:DNA, Neoplasm; D001622:Betaine; C512478:EGFR protein, human; D066246:ErbB Receptors; D005990:Glycerol; D004121:Dimethyl Sulfoxide
Additives; Betaine; DMSO; EGFR; Glycerol; Optimization; PCR