UC San Diego

We present a technique based on Fluorescent Resonance Energy Transfer (FRET) mechanism that enables single- molecule sequencing with a minute amount of starting cells or tissues. During DNA polymerase catalyzed nucleotide incorporation, a donor fluorophore conjugated onto the polymerase acts as a transition state for a highly localized transfer of energy to a recipient fluorophore on a target deoxyribonucleotide triphosphate (dNTP). This enables local signal amplification in four target wavelengths eliminating the need for DNA amplification through PCR or waveguide-mediated signal boosting. The donor particle is stimulated using a 532nm laser in a total internal reflection microscope (TIRFM) setup that reduces background noise by using evanescently propagating waves to interact only within 100nm of the surface of the glass substrate. The sample can be prepped using fluidics in low concentrations (< 100pM) and is attached onto a biotin-streptavidin functionalized surface. Since the fluorescent dNTPs are not end-terminated, all templates exposed to the dNTP solution will sequence, regardless if they are being observed. Our solution is a microfluidic polydimethylsiloxane (PDMS) device that is capable of individually addressing isolated sequencing sites. We demonstrate the viability of the complete system to provide accurate short template sequencing and provide a proof of principle for DNA methylation detection and long- template sequencing on our next-generation protocols and devices