Novel methylation biomarker panel for the early detection of pancreatic cancer.

Purpose: Pancreatic cancer is the fourth leading cause of cancer deaths and there currently is no reliable modality for the early detection of this disease. Here, we identify cancer-specific promoter DNA methylation of BNC1 and ADAMTS1 as a promising biomarker detection strategy meriting investigation in pancreatic cancer.

Experimental Design: We used a genome-wide pharmacologic transcriptome approach to identify novel cancer-specific DNA methylation alterations in pancreatic cancer cell lines.

DNA methylation detection using MS-qFRET, a quantum dot-based nanoassay.

Detection of aberrant promoter hypermethylation of tumor suppressor genes can be used as a prognostic or predictive marker for carcinogenesis. Since epigenetic modifying agents are FDA approved for treatment of patients with myelodysplastic syndrome, laboratory correlative tools to monitor response to this targeted therapy are important. Methylation specific quantum dot fluorescence resonance energy transfer (MS-qFRET) is a nanotechnology assay that enables the detection of methylation and its changes in a sensitive, quantifiable manner.

Single-tube analysis of DNA methylation with silica superparamagnetic beads.

Background: DNA promoter methylation is a signature for the silencing of tumor suppressor genes. Most widely used methods to detect DNA methylation involve 3 separate, independent processes: DNA extraction, bisulfite conversion, and methylation detection via a PCR method, such as methylation-specific PCR (MSP). This method includes many disconnected steps with associated losses of material, potentially reducing the analytical sensitivity required for analysis of challenging clinical samples.

MS-qFRET: a quantum dot-based method for analysis of DNA methylation.

DNA methylation contributes to carcinogenesis by silencing key tumor suppressor genes. Here we report an ultrasensitive and reliable nanotechnology assay, MS-qFRET, for detection and quantification of DNA methylation. Bisulfite-modified DNA is subjected to PCR amplification with primers that would differentiate between methylated and unmethylated DNA.

Tunable blinking kinetics of cy5 for precise DNA quantification and single-nucleotide difference detection.

Fluorescence correlation spectroscopy (FCS) can resolve the intrinsic fast-blinking kinetics (FBKs) of fluorescent molecules that occur on the order of microseconds. These FBKs can be heavily influenced by the microenvironments in which the fluorescent molecules are contained. In this work, FCS is used to monitor the dynamics of fluorescence emission from Cy5 labeled on DNA probes.

Intercalating dye as an acceptor in quantum-dot-mediated FRET.

Fluorescence resonance energy transfer (FRET) is a popular tool to study intermolecular distances and characterize structural or conformational changes of biological macromolecules. We investigate a novel inorganic/organic FRET pair with quantum dots (QDs) as donors and DNA intercalating dyes, BOBO-3, as acceptors by using DNA as a linker. Typically, FRET efficiency increases with the number of stained DNA linked to a QD.

Quantification of low concentrations of DNA using single molecule detection and velocity measurement in a microchannel.

We present a novel method for quantifying low concentrations of DNA based on single molecule detection (SMD) for molecular counting and flow measurements inside a microchannel. A custom confocal fluorescence spectroscopic system is implemented to detect fluorescent bursts emitted from stained DNA molecules. Measurements are made one molecule at a time as they flow through a femtoliter-sized laser focal probe.