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
A general procedure for the 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-mediated electrooxidation of primary and secondary alcohols modified for application in a microfluidic electrolytic cell is described. The electrocatalytic system utilises a buffered aqueous tert-butanol reaction medium, which operates effectively without the requirement for additional electrolyte, providing a mild protocol for the oxidation of alcohols to aldehydes and ketones at ambient temperature on a laboratory scale. Optimisation of the process is discussed along with the oxidation of 15 representative alcohols.
Subject(s)
Alcohols/chemistry , Cyclic N-Oxides/chemistry , Electrolysis/instrumentation , Green Chemistry Technology/instrumentation , Microfluidic Analytical Techniques/methods , Electrochemistry , Hydrogen-Ion Concentration , Oxidation-Reduction , TemperatureABSTRACT
High-quality films of bismuth antimony telluride were synthesized by electrodeposition from nitric acid electroplating baths. The influence of a surfactant, sodium ligninsulfonate, on the structure, morphology, stoichiometry, and homogeneity of the deposited films has been investigated. It was found that addition of this particular surfactant significantly improved the microstructural properties as well as homogeneity of the films with a significant improvement in the thermoelectric properties over those deposited in the absence of surfactant. A detailed microprobe analysis of the deposited films yielded a stoichiometric composition of Bi(0.35)Sb(1.33)Te(3) for the films electrodeposited in the absence of surfactant and a stoichiometry of Bi(0.32)Sb(1.33)Te(3) for films deposited in the presence of surfactant.
