优化 PCR 条件以扩增富含 GC 的 EGFR 启动子序列。
Optimization of PCR conditions for amplification of GC-Rich EGFR promoter sequence.
机构信息
Faculty of Medical Sciences, University of Kragujevac, Kragujevac, Serbia; Faculty of Science, Institute of Biology and Ecology, University of Kragujevac, Kragujevac, Serbia.
出版信息
J Clin Lab Anal. 2013 Nov;27(6):487-93. doi: 10.1002/jcla.21632.
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.
背景
聚合酶链反应（PCR）是一种极其灵敏的方法，通常需要进行优化，特别是在需要扩增困难模板时。本研究的目的是优化扩增表皮生长因子受体（EGFR）启动子序列的 PCR 条件，该序列具有极高的鸟嘌呤-胞嘧啶（GC）含量，以检测单核苷酸多态性 -216G>T 和 -191C>A。
方法
用于扩增的基因组 DNA 从福尔马林固定石蜡包埋的肺肿瘤组织中提取，并通过琼脂糖凝胶电泳检测 PCR 产物。
结果
结果表明，成功扩增需要添加 5%二甲亚砜（DMSO），以及 PCR 反应中至少 2μg/ml 的 DNA 浓度。由于 GC 含量高，最佳退火温度比计算值高 7°C，而合适的 MgCl2 浓度范围为 1.5 至 2.0 mM。
结论
总之，EGFR 启动子区域是一个难以进行 PCR 的靶标，但在存在 DMSO 和可接受浓度的 DNA 模板的情况下，可以通过优化 MgCl2 浓度和退火温度来进行扩增。
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