DNA methylation density analysis via fluorescent dye incorporation
Authors: Keeley Brian P. , Bailey Vasudev J. , Yin Chao , Passlack Katrin , Stark Alejandro , Carraway Hetty E. , Baylin Stephen B. , Herman James G. , Wang Tza-Huei “Jeff” 
Published: May 2015

Abstract
Background: Aberrant DNA methylation of cytosine nucleotides within the promoter region of tumor suppressor genes provides an epigenetic mechanism of transcriptional control associated with carcinogenesis. Thus, DNA methylation analysis holds clinical potential for early detection and treatment of cancer. Both qualitative detection methods such as methylation specific PCR (MSP), and quantitative methods like MethyLight and Ms-SNuPE analyze a proportion of alleles only when they are heavily methylated at primer/probe sequences. Bisulfite sequencing can be useful to understand methylation density but is not often used quantitatively due to the labor intensive sequencing of multiple plasmid clones.
Methods and Results: Methylation through Fluorescence of a Single Strand (Methyl-FloSS) establishes a simple detection method for the quantitative analysis of methylation density. Methyl-FloSS begins with the bisulfite treatment of genomic DNA, followed by PCR amplification with primers that are independent of methylation status. Amplification is carried out with labeled nucleotides (Cy5-dCTP) and a 5’ phosphate conjugated primer. Enzymatic digestion is then directed toward the reverse strand containing the phosphate primer, allowing investigation of the remaining strand wherein Cy5 fluorophores are incorporated into the positions of methylated cytosines. Therefore, the methylation density is proportional to the measured fluorescence intensity of the single stranded amplicon.
A fluorescence density score (FDS) is calculated to quantify the methylation density. By including a positive and negative control and assigning a FDS of 1 and 0 respectfully, each sample receives a FDS score based upon the normalized fluorescence intensity and DNA concentration. To demonstrate the applicability of Methyl-FloSS, we observed the in vitro effect of 5-aza-2’-deoxycytidine (DAC) on the methylated p15INK4B promoter region in KG-1a cell line. Cells were harvested after 72 hours of incubation with 0 nM, 10 nM, 100 nM, and 1 µM of DAC. Our results demonstrated a dose-dependent decrease in p15INK4B promoter methylation density as measured by decreasing FDS, which is consistent with prior MSP analysis.
Demonstrating the clinical significance of the Methyl-FloSS, blood samples from 24 acute myeloid leukemia (AML) patients were comparatively analyzed by both MSP categorization and density analysis. One patient was considered methylated for p15INK4B by MSP, but density analysis measured a low FDS. This result indicates that the presence of methylated cytosines located within the primer sequences of MSP may categorize the patient as methylated; however, the methylation density of the promoter region is comparable with unmethylated controls. Overall, FDS scores allowed for a clear segregation between methylated and unmethylated samples, featuring greater resolution by fluorescence measurement.
Conclusion: The enhanced detection of methylation density by Methyl-FloSS provides a convenient, quantitative analysis of promoter methylation status and is not biased by primer/probe sequences. Quantification through FDS will provide greater insight and analysis into epigenetic alterations and therapies. In addition, Methyl-FloSS avoids radioactive labeling and can be easily implemented in a multi-well format for high throughput analysis.