A real-time PCR assay for DNA-methylation using methylation-specific blockers

Abstract

DNA methylation-based biomarkers have been discovered that could potentially be used for the diagnosis of cancer by detection of circulating, tumor-derived DNA in bodily fluids. Any methylation detection assay that would be applied to these samples must be capable of detecting small amounts of tumor DNA in the presence of background normal DNA. We have developed a real-time PCR assay, called HeavyMethyl, that is well suited for this application. HeavyMethyl uses methylation-specific oligonucleotide blockers and a methylation-specific probe to achieve methylation-specific amplification and detection. We tested the assays on unmethylated and artificially methylated DNA in order to determine the limit of detection. After careful optimization, our glutathione-S-transferase pi1 and Calcitonin assays can amplify as little as 30 and 60 pg of methylated DNA, respectively, and neither assay amplifies unmethylated DNA. The Calcitonin assay showed a highly significant methylation difference between normal colon and colon adenocarcinomas, and methylation was also detected in serum DNA from colon cancer patients. These assays show that HeavyMethyl technology can be successfully employed for the analysis of very low concentrations of methylated DNA, e.g. in serum of patients with tumors.

Evaluation of control transcripts in real-time RT-PCR expression analysis during maritime pine embryogenesis

“…The high sensitivity, accuracy and reproducibility of the technique support the numerous applications of real-time RT-PCR (Kang et al 2000;Mackay et al 2002;Cottrell et al 2004;Johnson et al 2004). When measuring RNA expression an important feature is the normalization between samples.…”

Precision and Performance Characteristics of Bisulfite Conversion and Real-Time PCR (MethyLight) for Quantitative DNA Methylation Analysis

“…Hence, a variety of quantitative assays to measure DNA methylation have been developed, including combined bisulfite restriction analysis, 14 restriction ligation-mediated polymerase chain reaction (PCR), 15 methylation-sensitive single nucleotide primer extension (Ms-SNuPE), 16 ion-pair reverse-phase high performance liquid chromatography, 17 denaturing high performance liquid chromatography, 18 pyrosequencing, 19 -21 MALDI-TOF, 22 and real-time PCR. 5,[23][24][25][26][27][28][29][30][31] Most of these methods use DNA treated with sodium bisulfite. Therefore, sodium bisulfite treatment is a critical step in the measurement of DNA methylation.…”

Screening for Colorectal Cancer Based on the Promoter Methylation Status of the Septin 9 Gene in Plasma Cell Free DNA

“…This test comprised magnetic particle based DNA isolation from 3.5 mLs plasma, bisulfite conversion using sodium bisulfite, repurification using magnetic particles, a duplex real-time PCR measuring actin beta as an internal DNA concentration control, and methylated SEPT9 promoter DNA as the target biomarker [28]. The PCR reaction is based on Heavy Methyl amplification using a blocker oligonucleotide to suppress amplification of non-methylated background DNA, combined with Methyl Light detection using a methylation specific detection probe [29]. Using this approach, the analytical sensitivity of the assay approached three genome equivalents per mL of plasma.…”