ABSTRACT
Despite remarkable progress in DNA sequencing technologies there remains a trade-off between short-read platforms, having limited ability to sequence homopolymers, repeated motifs or long-range structural variation, and long-read platforms, which tend to have lower accuracy and/or throughput. Moreover, current methods do not allow direct readout of epigenetic modifications from a single read. With the aim of addressing these limitations, we have developed an optical electrowetting sequencing platform that uses step-wise nucleotide triphosphate (dNTP) release, capture and detection in microdroplets from single DNA molecules. Each microdroplet serves as a reaction vessel that identifies an individual dNTP based on a robust fluorescence signal, with the detection chemistry extended to enable detection of 5-methylcytosine. Our platform uses small reagent volumes and inexpensive equipment, paving the way to cost-effective single-molecule DNA sequencing, capable of handling widely varying GC-bias, and demonstrating direct detection of epigenetic modifications.
Subject(s)
DNA/genetics , High-Throughput Nucleotide Sequencing , Sequence Analysis, DNA/methods , Single Molecule Imaging , Base Composition/genetics , Humans , Nanotechnology , Nucleotides/geneticsABSTRACT
A new approach to single-molecule DNA sequencing in which dNTPs, released by pyrophosphorolysis from the strand to be sequenced, are captured in microdroplets and read directly could have substantial advantages over current sequence-by-synthesis methods; however, there is no existing method sensitive enough to detect a single nucleotide in a microdroplet. We have developed a method for dNTP detection based on an enzymatic two-stage reaction which produces a robust fluorescent signal that is easy to detect and process. By taking advantage of the inherent specificity of DNA polymerases and ligases, coupled with volume restriction in microdroplets, this method allows us to simultaneously detect the presence of and distinguish between, the four natural dNTPs at the single-molecule level, with negligible cross-talk.
Subject(s)
Deoxyribonucleotides/analysis , High-Throughput Nucleotide Sequencing/methods , Sequence Analysis, DNA/methods , DNA-Directed DNA Polymerase/metabolism , Deoxyribonucleosides/chemistry , Deoxyribonucleotides/chemistry , Limit of Detection , Microscopy, Fluorescence , Oligodeoxyribonucleotides/biosynthesis , Oligodeoxyribonucleotides/chemistry , Sensitivity and SpecificityABSTRACT
Self-assembled mesoporous structures with well-ordered nanoscale channels could be used in applications such as molecular separation, nano-optics, molecular electronics, nanomedicine and catalysis. However, the domain sizes that can be created in such systems are limited by our lack of a detailed understanding of the relevant growth processes. Here we report the real-time observation of domain growth in the self-assembly of silica nanochannels using fluorescence polarization imaging and atomic force microscopy. We show that transient lamellar structures precede the formation of hexagonal layers, and that the layer growth follows two distinct pathways. In addition, the domains are grown on a mesoporous film substrate, which acts as a sieve and allows control of the delivery of the reactive species. We use these insights and capabilities to grow layers of well-ordered silica nanochannels with domain sizes of up to â¼0.3 mm.
Subject(s)
Nanostructures/chemistry , Nanostructures/ultrastructure , Silicon Dioxide/chemistry , Diffusion , Fluorescent Dyes , Kinetics , Microscopy, Atomic Force , Microscopy, Fluorescence , PorosityABSTRACT
The influence of an acetamido group in directing the preferred choice of hydration sites in glucosamine and a consequent extension of the working rules governing regioselective hydration and conformational choice, have been revealed through comparisons between the conformations and structures of "free" and multiply hydrated phenyl N-acetyl-beta-D-glucosamine (betapGlcNAc) and phenyl beta-D-glucopyranoside (betapGlc), isolated in the gas phase at low temperatures. The structures have been assigned through infrared ion depletion spectroscopy conducted in a supersonic jet expansion, coupled with computational methods. The acetamido motif provides a hydration focus that overwhelms the directing role of the hydroxymethyl group; in multiply hydrated betapGlcNAc the water molecules are all located around the acetamido motif, on the "axial" faces of the pyranose ring rather than around its edge, despite the equatorial disposition of all the hydrophilic groups in the ring. The striking and unprecedented role of the C-2 acetamido group in controlling hydration structures may, in part, explain the differing and widespread roles of GlcNAc, and perhaps GalNAc, in nature.
