Optimal primer design using the novel primer design program: MSPprimer provides accurate methylation analysis of the ATM promoter.
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.
Brandes
J C
JC
Cancer Biology Program, Department of Oncology, The Sidney Kimmel Comprehensive Cancer, Center at Johns Hopkins, Baltimore, MD 21231-1000, USA.
Carraway
H
H
Herman
J G
JG
D000465:Algorithms; D064007:Ataxia Telangiectasia Mutated Proteins; D001483:Base Sequence; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000378:metabolism*; D045744:Cell Line, Tumor; D019295:Computational Biology; D019175:DNA Methylation; D017931:DNA Primers / Q000138:chemical synthesis*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D045325:HCT116 Cells; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism*; D012984:Software; D025521:Tumor Suppressor Proteins / Q000235:genetics* / Q000378:metabolism*
D018797:Cell Cycle Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D025521:Tumor Suppressor Proteins; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases