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Clinical Chemistry 53: 2119-2127, 2007. First published October 25, 2007; 10.1373/clinchem.2007.094011
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Right arrow Cancer Diagnostics (since 2002)
(Clinical Chemistry. 2007;53:2119-2127.)
© 2007 American Association for Clinical Chemistry, Inc.

Cancer Diagnostics

MS-FLAG, a Novel Real-Time Signal Generation Method for Methylation-Specific PCR

Cinzia Bonanno1, Erlet Shehi2, Daniel Adlerstein2,a and G. Mike Makrigiorgos3

1 University of Milano Bicocca, Milano, Italy.
2 DiaSorin SpA, Saluggia (VC), Italy.
3 Dana Farber-Brigham and Women’s Cancer Center, Harvard Medical School, Boston, MA.

aAddress correspondence to this author at: Diasorin SpA, Viale Pasteur 10, 20014 Nerviano (MI), Italy. Fax 0039-03311547; e-mail daniel.adlerstein{at}diasorin.it.

Background: Aberrant promoter methylation is a major mechanism for silencing tumor suppressor genes in cancer. Detection of hypermethylation is used as a molecular marker for early cancer diagnosis, as a prognostic index, or to define therapeutic targets for reversion of aberrant methylation. We report on a novel signal generation technology for real-time PCR to detect gene promoter methylation.

Methods: FLAG (fluorescent amplicon generation) is a homogeneous signal generation technology based on the exceptionally thermostable endonuclease PspGI. FLAG provides real-time signal generation during PCR by PspGI-mediated cleavage of quenched fluorophores at the 5' end of double-stranded PCR products. Methylation-specific PCR (MSP) applied on bisulfite-treated DNA was adapted to a real-time format (methylation-specific FLAG; MS-FLAG) for quantifying methylation in the promoter of CDKN2A (p16), GATA5, and RASSF1. We validated MS-FLAG on plasmids and genomic DNA with known methylation status and applied it to detection of methylation in a limited number of clinical samples. We also conducted bisulfite sequencing on these samples.

Results: Real-time PCR results obtained via MS-FLAG agreed with results obtained via conventional, gel-based MSP. The new technology showed high specificity, sensitivity (2–3 plasmid copies), and selectivity (0.01% of methylated DNA) on control samples. It enabled correct prediction of the methylation status of all 3 gene promoters in 21 lung adenocarcinoma samples, as confirmed by bisulfite sequencing. We also developed a multiplex MS-FLAG assay for GATA5 and RASSF1 promoters.

Conclusion: MS-FLAG provides a new, quantitative, high-throughput method for detecting gene promoter methylation and is a convenient alternative to agarose gel-based MSP for screening methylation. In addition to methylation, FLAG-based real-time signal generation may have broad applications in DNA diagnostics.






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