Evaluating the sensitivity of hybridization-based epigenotyping using a methyl binding domain protein

Abstract

Hypermethylation of CpG islands in gene promoter regions has been shown to be a predictive biomarker for certain diseases. Most current methods for methylation profiling are not well-suited for clinical analysis. Here, we report the development of an inexpensive device and an epigenotyping assay with a format conducive to multiplexed analysis.

Fig. 1

Methylated and hydrazide functionalized probe ssDNA oligos are spotted onto activated agarose coated slides with subsequent blocking using 1% w/v BSA. Target ssDNA oligos for analysis are hybridized to the sequence-specific probes on the biochip. A biotinylated MBD protein is then added to bind methylated CpG dinucleotides followed by streptavidin-Cy3 for fluorescence or streptavidin-eosin for polymerization readout.

Fig. 2

(a) Biochips were spotted with capture probe ssDNA oligos having two (omm), one (omo), or no (ooo) methylated CpGs in order to epigenotype the target oligos. Unmethylated ssDNA served as the negative control. (b) Schematic representation of the area within each group of spots following hybridization with 100 nM doubly methylated (omm) target ssDNA and detection using the scheme shown in Fig. 1. Fluorescence (c) and colorimetric (d) readout of MBD binding to methylated DNA.

Fig. 3

A microfluidic device was mated to the biochip. (a) Diagram of the microfluidic channel etched in PDMS. (b) Supporting glass was added to the unetched side of the microfluidic device, and it was clamped to the biochip. Inlet and outlet tubing was connected to each end of the channel, fed into a microcentrifuge tube reservoir, and passed through a peristaltic pump for recirculation. (c) Comparison of the signal from static MBD binding to doubly (omm) and singly (omo) methylated probe ssDNA hybridized with either 100, 10, or 1 nM doubly (omm) methylated target ssDNA under static or microfluidic conditions. Microfluidic hybridization increases the signal from MBD binding by approximately one-third. (d) Comparison of the signal from MBD binding under static or 100 µL/min recycling conditions to doubly and singly methylated duplexes. The microfluidic device does not improve signal due to a weak dependence of MBD binding on flow rate and a partial hybridization reaction limit.

Fig. 4

The limit of quantification (defined as the minimum concentration producing a signal greater than that from the unmethylated (ooo) probe mean plus 10 SDs) for doubly (omm) and singly (omo) symmetrically methylated DNA duplexes was determined to be 0.3 nM and 1 nM, respectively. Each sample was on-chip hybridized with the stated concentration with omm target ssDNA and detected with 40 µg/mL MBD followed by streptavidin-Cy3 labeling.