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Background: Aberrant DNA methylation of gene promoters and the associated silencing of tumor suppressor genes are recognized as mechanisms contributing to tumor development. Therefore, detection of promoter hypermethylation is becoming important for diagnosis, prognosis, and aiding the design of cancer therapies. We describe a novel isothermal method for the detection of DNA hypermethylation., Methods: Methylation-specific loop-mediated isothermal amplification (MS-LAMP) is a novel adaptation of LAMP. MS-LAMP was used for the highly specific detection of hypermethylated CpGs in the promoters of the CDKN2A [cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4)], GATA5 (GATA binding protein 5), and DAPK1 (death-associated protein kinase 1) genes. The reactions occurred under isothermal conditions with 3 primer sets specific for methylated promoters. Both turbidimetry and fluorescence were used for detection. The MS-LAMP assay was validated with bisulfite-treated plasmid and genomic DNA controls of known methylation status and was applied to detect hypermethylation in 18 clinical tumor samples. A multiplex MS-LAMP for CDKN2A, GATA5, and DAPK1 was also validated with the aid of synthetic positive and negative controls., Results: The MS-LAMP assay showed high specificity with plasmid and genomic DNA targets in reactions carried out in <1 h. The assay had a detection limit of approximately 30 copies of methylated target sequence and a selectivity of 0.5% methylated DNA in a mixture with unmethylated DNA. Compared with methylation-specific PCR, the MS-LAMP assay detected lower rates of methylation in lung adenocarcinoma samples. Simultaneous multiplex detection of hypermethylation in the 3 targets (CDKN2A, GATA5, and DAPK1) was readily achieved with the MS-LAMP assay in both the turbidimetric and fluorescence detection formats., Conclusions: MS-LAMP provides a highly specific isothermal method for methylation detection and is well suited for multiplex approaches.

Background: Monitoring the human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), or varicella-zoster virus (VZV) viral load is an important factor in the management of immunosuppressed patients, such as recipients of solid-organ or bone marrow transplants. The advent of real-time PCR technologies has prompted the widespread development of quantitative PCR assays for the detection of viral loads and other diagnostic purposes., Methods: The fluorescent amplicon generation (FLAG) technology uses the PspGI restriction enzyme to monitor PCR product generation. We modified the FLAG technology by introducing an accessory oligonucleotide "anchor" that stabilizes the binding of the forward primer to the target sequence (a-FLAG). We developed assays for HCMV, EBV, and VZV that incorporated an internal amplification-control reaction to validate negative results and extensively analyzed the performance of the HCMV a-FLAG assay., Results: The 3 assays performed similarly with respect to reaction efficiency and linear range. Compared with a commercially available kit, the HCMV a-FLAG assay results showed good correlation with calculated concentrations (r = 0.9617), excellent diagnostic sensitivity and specificity (99% and 95%, respectively), and similar values for the linear range (1-10(7) copies/microL), analytical sensitivity (0.420 copies/microL), and intra- and interassay imprecision., Conclusions: The a-FLAG assay is an alternative real-time PCR technology suitable for detecting and quantifying target-DNA sequences. For clinical applications such as the measurement of viral load, a-FLAG assays provide multiplex capability, internal amplification control, and high diagnostic sensitivity and specificity.

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.

Real-time signal generation methods for detection and characterization of low-abundance mutations in genomic DNA are powerful tools for cancer diagnosis and prognosis. Mutations in codon 12 of the oncogene KRAS, for example, are frequently found in several types of human cancers. We have developed a novel real-time PCR technology, FLAG (FLuorescent Amplicon Generation) and adapted it for simultaneously (i) amplifying mutated codon 12 KRAS sequences, (ii) monitoring in real-time the amplification and (iii) genotyping the exact nucleotide alteration. FLAG utilizes the exceptionally thermostable endonuclease PspGI for real-time signal generation by cleavage of quenched fluorophores from the 5'-end of the PCR products and, concurrently, for selecting KRAS mutations over wild type. By including peptide-nucleic-acid probes in the reaction, simultaneous genotyping is achieved that circumvents the requirement for sequencing. FLAG enables high-throughput, closed-tube KRAS mutation detection down to approximately 0.1% mutant-to-wild type. The assay was validated on model systems and compared with allele-specific PCR sequencing for screening 27 cancer specimens. Diverse applications of FLAG for real-time PCR or genotyping applications in cancer, virology or infectious diseases are envisioned.

Background: Genotyping methods that reveal single-nucleotide differences are useful for a wide range of applications. We used digestion of 3-way DNA junctions in a novel technology, OneCutEventAmplificatioN (OCEAN) that allows sequence-specific signal generation and amplification. We combined OCEAN with peptide-nucleic-acid (PNA)-based variant enrichment to detect and simultaneously genotype v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) codon 12 sequence variants in human tissue specimens., Materials and Methods: We analyzed KRAS codon 12 sequence variants in 106 lung cancer surgical specimens. We conducted a PNA-PCR reaction that suppresses wild-type KRAS amplification and genotyped the product with a set of OCEAN reactions carried out in fluorescence microplate format. The isothermal OCEAN assay enabled a 3-way DNA junction to form between the specific target nucleic acid, a fluorescently labeled "amplifier", and an "anchor". The amplifier-anchor contact contains the recognition site for a restriction enzyme. Digestion produces a cleaved amplifier and generation of a fluorescent signal. The cleaved amplifier dissociates from the 3-way DNA junction, allowing a new amplifier to bind and propagate the reaction., Results: The system detected and genotyped KRAS sequence variants down to approximately 0.3% variant-to-wild-type alleles. PNA-PCR/OCEAN had a concordance rate with PNA-PCR/sequencing of 93% to 98%, depending on the exact implementation. Concordance rate with restriction endonuclease-mediated selective-PCR/sequencing was 89%., Conclusion: OCEAN is a practical and low-cost novel technology for sequence-specific signal generation. Reliable analysis of KRAS sequence alterations in human specimens circumvents the requirement for sequencing. Application is expected in genotyping KRAS codon 12 sequence variants in surgical specimens or in bodily fluids, as well as single-base variations and sequence alterations in other genes.