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

Abstract

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 (methylationspecific 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

Anchor-Based Fluorescent Amplicon Generation Assays (FLAG) for Real-Time Measurement of Human Cytomegalovirus, Epstein–Barr Virus, and Varicella-Zoster Virus Viral Loads

“…This and previous work (12,13 ) indicate that the FLAG principle could be adapted to develop real-time assays for a wide range of clinical applications.…”

“…The FLAG system uses the thermostable restriction enzyme PspGI to cleave a fluorophore-quencher pair that is present at the end of double-stranded amplification products (12,13 ). In this work, we introduced an accessory oligonucleotide ("anchor") to facilitate the annealing of the forward primer (see Supplemental Figure 1 and Supplemental Table 1 in the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol54/issue11), which carries at the 5Ј end an 11-base sequence tag containing a fluorophore-quencher pair separated by a restricted site for PspGI.…”

“…Accordingly, highly reliable real-time PCR assays must be used to correctly identify samples that are positive and negative for these viruses (8,9 ). Fluorescent amplicon generation (FLAG) was recently described as a primer-based signal-generation technology for detecting mutations (12 ) and DNA hypermethylation (13 ). In the present study, we have included an accessory oligonucleotide (an "anchor") in the FLAG assay (a-FLAG).…”

Methylation-Specific Loop-Mediated Isothermal Amplification for Detecting Hypermethylated DNA in Simplex and Multiplex Formats

“…In contrast, techniques that use only 2 primers, such as MSP or MS-FLAG, often score a higher number of samples as positives. Similarly, MethyLight, a methylationdetection technology that probes 3 target regions, may also demonstrate a lower scoring rate of methylationpositive clinical samples compared with the MSP methodology (24 ). In designing our MS-LAMP primer sets, we maximized the specificity for methylated DNA after bisulfite conversion by appropriate positioning of the primers on the target DNA to include as many CpG dinucleotides per primer as possible (mean of 2-3 CpGs per primer region, with some being close to the 3Ј ends).…”

“…Moreover, some PCR-based approaches rely on the use of primer sets designed specifically to recognize and amplify only methylated (or, alternatively, unmethylated) DNA sequences due to the bisulfite conversion of the target DNA. PCR amplification can be detected at the end of the reaction via gel electrophoresis [i.e., methylation-specific PCR (MSP) 5 ] (21 ) or by real-time PCR techniques [MethyLight and methylation-specific fluorescent amplicon generation (MS-FLAG)] (22)(23)(24).…”