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

J. C. Brandes, H. Carraway, J. G. Herman

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)6229-6237
Number of pages9
JournalOncogene
Volume26
Issue number42
DOIs
StatePublished - Sep 13 2007

Fingerprint

Methylation
Non-Small Cell Lung Carcinoma
Polymerase Chain Reaction
Breast Neoplasms
Gene Silencing
Thermodynamics
DNA
Incidence
hydrogen sulfite

Keywords

  • ATM
  • Breast cancer
  • DNA methylation
  • Lung cancer
  • Methylation Specific PCR

ASJC Scopus subject areas

  • Molecular Biology
  • Cancer Research
  • Genetics

Cite this

Optimal primer design using the novel primer design program : MSPprimer provides accurate methylation analysis of the ATM promoter. / Brandes, J. C.; Carraway, H.; Herman, J. G.

In: Oncogene, Vol. 26, No. 42, 13.09.2007, p. 6229-6237.

Research output: Contribution to journalArticle

Brandes, J. C. ; Carraway, H. ; Herman, J. G. / Optimal primer design using the novel primer design program : MSPprimer provides accurate methylation analysis of the ATM promoter. In: Oncogene. 2007 ; Vol. 26, No. 42. pp. 6229-6237.
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