底漆(化妆品)
生物
甲基化
亚硫酸氢盐测序
DNA甲基化
亚硫酸氢盐
多重聚合酶链反应
分子生物学
聚合酶链反应
多路复用
计算生物学
基因
遗传学
基因表达
化学
有机化学
作者
Johann C. Brandes,Hetty E. Carraway,James G. Herman
出处
期刊:Oncogene
[Springer Nature]
日期:2007-03-26
卷期号:26 (42): 6229-6237
被引量:69
标识
DOI:10.1038/sj.onc.1210433
摘要
Methylation-specific polymerase chain reaction (PCR) (MSP) is frequently used to study gene silencing by promoter hypermethylation. However, non-specific primer design can lead to false-positive detection of methylation. We present a novel, web-based algorithm for the design of primers for bisulfite-PCRs (MSP, sequencing, COBRA and multiplex-MSP), allowing the determination of a specificity score, which is based on the thermodynamic characteristics of the primer 3′-end. PCR amplification with primers not reaching a high specificity score can result in false-positive findings. We used MSPprimer to design MSP primers for analysis of the ATM promoter. In 37 non-small cell lung cancer (NSCLC) samples and 43 breast cancer samples no promoter methylation was detected. Conversely, published MSP primers not reaching the required specificity score led to non-specific amplification of DNA not converted by bisulfite. The result was a false-positive incidence of ATM promoter methylation of 24% in NSCLC and 48% in breast cancers, similar to published studies. This highlights the critical need for specific primer design for MSP. MSPprimer is a convenient tool to achieve this goal, which is available free of charge to the scientific community.
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