التفاصيل البيبلوغرافية
| العنوان: |
Optimal primer design using the novel primer design program: MSPprimer provides accurate methylation analysis of the ATM promoter. |
| المؤلفون: |
Brandes, J. C.1, Carraway, H.1, Herman, J. G.1 hermanji@jhmi.edu |
| المصدر: |
Oncogene. 9/13/2007, Vol. 26 Issue 42, p6229-6237. 9p. 1 Black and White Photograph, 3 Diagrams, 1 Chart. |
| مصطلحات موضوعية: |
*METHYLATION, *POLYMERASE chain reaction, *GENE silencing, *BREAST cancer, *SMALL cell lung cancer, *GENETIC regulation |
| مستخلص: |
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.Oncogene (2007) 26, 6229–6237; doi:10.1038/sj.onc.1210433; published online 26 March 2007 [ABSTRACT FROM AUTHOR] |
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