MS-FLAG, a novel real-time signal generation method for methylation-specific PCR.
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.


CONCLUSIONS
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.
Bonanno
Cinzia
C
University of Milano Bicocca, Milano, Italy.
Shehi
Erlet
E
Adlerstein
Daniel
D
Makrigiorgos
G Mike
GM
D000230:Adenocarcinoma / Q000235:genetics; D018899:CpG Islands; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics*; D019175:DNA Methylation; D005453:Fluorescence; D050992:GATA5 Transcription Factor / Q000235:genetics*; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic; D012680:Sensitivity and Specificity; D013447:Sulfites; D025521:Tumor Suppressor Proteins / Q000235:genetics*
D019941:Cyclin-Dependent Kinase Inhibitor p16; D050992:GATA5 Transcription Factor; C494755:GATA5 protein, human; C411762:RASSF1 protein, human; D013447:Sulfites; D025521:Tumor Suppressor Proteins; C009279:sodium bisulfite