Optimization of PCR Conditions for Amplification of GC-Rich EGFR Promoter Sequence
2013
Аутори
Obradović, JasminaJurisić, Vladimir
Tošić, Nataša
Mrdjanović, Jasminka
Perin, Branislav
Pavlović, Sonja
Đorđević, Nataša
Чланак у часопису (Објављена верзија)
Метаподаци
Приказ свих података о документуАпстракт
BackgroundPolymerase 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 gt T and -191C gt A. MethodsGenomic DNA used for amplification was extracted from formalin-fixed paraffin-embedded lung tumor tissue and PCR products were detected by agarose gel electrophoresis. ResultsResults 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 degrees C higher than calculated, while adequate MgCl2 concentration ranged from 1.5 to 2.0 mM. ConclusionIn conclusion, EGFR promoter re...gion 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.
Кључне речи:
PCR / optimization / genotyping / GC-rich / EGFRИзвор:
Journal of Clinical Laboratory Analysis, 2013, 27, 6, 487-493Издавач:
- Wiley, Hoboken
Финансирање / пројекти:
- Молекулске, биохемијске и имунолошке анализе у дијагностици тумора (RS-MESTD-Basic Research (BR or ON)-175056)
DOI: 10.1002/jcla.21632
ISSN: 0887-8013
PubMed: 24218132
WoS: 000326890600011
Scopus: 2-s2.0-84887379115
Институција/група
Institut za molekularnu genetiku i genetičko inženjerstvoTY - JOUR AU - Obradović, Jasmina AU - Jurisić, Vladimir AU - Tošić, Nataša AU - Mrdjanović, Jasminka AU - Perin, Branislav AU - Pavlović, Sonja AU - Đorđević, Nataša PY - 2013 UR - https://imagine.imgge.bg.ac.rs/handle/123456789/666 AB - BackgroundPolymerase 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 gt T and -191C gt A. MethodsGenomic DNA used for amplification was extracted from formalin-fixed paraffin-embedded lung tumor tissue and PCR products were detected by agarose gel electrophoresis. ResultsResults 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 degrees C higher than calculated, while adequate MgCl2 concentration ranged from 1.5 to 2.0 mM. ConclusionIn 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. PB - Wiley, Hoboken T2 - Journal of Clinical Laboratory Analysis T1 - Optimization of PCR Conditions for Amplification of GC-Rich EGFR Promoter Sequence EP - 493 IS - 6 SP - 487 VL - 27 DO - 10.1002/jcla.21632 ER -
@article{ author = "Obradović, Jasmina and Jurisić, Vladimir and Tošić, Nataša and Mrdjanović, Jasminka and Perin, Branislav and Pavlović, Sonja and Đorđević, Nataša", year = "2013", abstract = "BackgroundPolymerase 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 gt T and -191C gt A. MethodsGenomic DNA used for amplification was extracted from formalin-fixed paraffin-embedded lung tumor tissue and PCR products were detected by agarose gel electrophoresis. ResultsResults 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 degrees C higher than calculated, while adequate MgCl2 concentration ranged from 1.5 to 2.0 mM. ConclusionIn 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.", publisher = "Wiley, Hoboken", journal = "Journal of Clinical Laboratory Analysis", title = "Optimization of PCR Conditions for Amplification of GC-Rich EGFR Promoter Sequence", pages = "493-487", number = "6", volume = "27", doi = "10.1002/jcla.21632" }
Obradović, J., Jurisić, V., Tošić, N., Mrdjanović, J., Perin, B., Pavlović, S.,& Đorđević, N.. (2013). Optimization of PCR Conditions for Amplification of GC-Rich EGFR Promoter Sequence. in Journal of Clinical Laboratory Analysis Wiley, Hoboken., 27(6), 487-493. https://doi.org/10.1002/jcla.21632
Obradović J, Jurisić V, Tošić N, Mrdjanović J, Perin B, Pavlović S, Đorđević N. Optimization of PCR Conditions for Amplification of GC-Rich EGFR Promoter Sequence. in Journal of Clinical Laboratory Analysis. 2013;27(6):487-493. doi:10.1002/jcla.21632 .
Obradović, Jasmina, Jurisić, Vladimir, Tošić, Nataša, Mrdjanović, Jasminka, Perin, Branislav, Pavlović, Sonja, Đorđević, Nataša, "Optimization of PCR Conditions for Amplification of GC-Rich EGFR Promoter Sequence" in Journal of Clinical Laboratory Analysis, 27, no. 6 (2013):487-493, https://doi.org/10.1002/jcla.21632 . .