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Optimal primer design using the novel primer design program: MSPprimer provides accurate methylation analysis of the ATM promoter

Oncogene volume 26, pages 6229–6237 (2007)Cite this article

A Corrigendum to this article was published on 11 October 2007

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.

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  1. Department of Oncology, Cancer Biology Program, The Sidney Kimmel Comprehensive Cancer, Center at Johns Hopkins, Baltimore, MD, USA

    J C Brandes, H Carraway & J G Herman

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  1. J C Brandes
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  2. H Carraway
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  3. J G Herman
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Correspondence to J G Herman.

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Brandes, J., Carraway, H. & Herman, J. Optimal primer design using the novel primer design program: MSPprimer provides accurate methylation analysis of the ATM promoter. Oncogene 26, 6229–6237 (2007). https://doi.org/10.1038/sj.onc.1210433

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