Optimal primer design using the novel primer ...

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Oncogene. 2007 Sep 13;26(42):6229-37. Epub 2007 Mar 26.
Optimal primer design using the novel primer design program: MSPprimer provides accurate methylation analysis of the ATM promoter.
Brandes JC,Carraway H,Herman JG.
Cancer Biology Program, Department of Oncology, The Sidney Kimmel Comprehensive Cancer, Center at Johns Hopkins, Baltimore, MD 21231-1000, USA.
Erratum in: Oncogene. 2007 Oct 11;26(46):6684.
 
Abstract
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.
PMID: 17384671 [PubMed - indexed for MEDLINE]
Publication Types, MeSH Terms, Substances
Publication Types:
Validation Studies
MeSH Terms:
AlgorithmsBase SequenceCell Cycle Proteins/genetics*Cell Cycle Proteins/metabolism*Cell Line, TumorComputational Biology*DNA Methylation*DNA Primers/chemical synthesis*DNA-Binding Proteins/genetics*DNA-Binding Proteins/metabolism*HCT116 CellsHumansMolecular Sequence DataPolymerase Chain Reaction*Promoter Regions, Genetic/physiology*Protein-Serine-Threonine Kinases/genetics*Protein-Serine-Threonine Kinases/metabolism*Software*Tumor Suppressor Proteins/genetics*Tumor Suppressor Proteins/metabolism*
Substances:
Cell Cycle ProteinsDNA PrimersDNA-Binding ProteinsTumor Suppressor Proteinsataxia telangiectasia mutated proteinProtein-Serine-Threonine Kinases
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Full Text Sources:
Nature Publishing GroupEBSCOOvid Technologies, Inc.ProQuest Information and LearningSwets Information Services
Medical:
Genetics Home Reference - ATM Gene - Genetics Home Reference
Supplemental Content

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