High throughput, quantitative DNA methylation screening using a quantum dot based nanotechnology assay

PL01

Background:
 The impact of detecting DNA methylation is profound, as it has been demonstrated that a larger number of tumor suppressor genes become inactivated by epigenetic silencing from DNA methylation than by mutations. Current methods of DNA methylation detection include qualitative methylation specific PCR (MSP) and quantitative real time PCR. We have demonstrated a nanotechnology assay that meets the needs for high throughput and ultra-sensitive DNA methylation analyses. The new technology will be likely to have a huge impact for both cancer diagnosis as well as assessing response to therapy.
 Methods:
 Our technology allows for performing multiple PCRs through a droplet-in-oil microfluidic PCR platform with single sample injection. It utilizes quantum dot (QD) nanosensors for the detection and quantification of DNA methylation. QDs serve as both a FRET donor as well as a nanoconcentrator to capture fluorescently labeled target amplicons which are generated through methylation specific PCR (MSP).
 Results:
 With our oil-in-droplet PCR platform, we were able to demonstrate valuable downscaling in reagent and sample consumption (800nL/ reaction). Our nanoassay allowed detecting as little as 15 pg of methylated DNA in the presence of a 10,000-fold excess of unmethylated alleles. We extended the analysis to clinical samples by detecting methylation for ASC/ TMS1 in low concentrations of DNA from human sputum without using nested PCR. The nanoassay featured low intrinsic background and high sensitivity with few PCR amplification cycles. We quantified methylation reversal with high resolution in pre and post bone marrow aspirate samples obtained from Myelodysplastic Syndrome (MDS) patients who were treated with epigenetic therapy using 5-azacytidine (Vidaza) and a histone deacetylase inhibitor (MS-275).
 Conclusion:
 Our QD-based nanoassay is fully compatible with the widely-used MSP. The use of an oil-in-droplet PCR system offers unprecedented ease in sample handling for a cost-effective multiplexed gene analysis. By simply end-labeling existing MSP primers and using commercially available QDs, our technique allows for quantitative, high-throughput and ultrasensitive methylation detection.