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Start Over Author "Bell NM" Journal nucleic acids research
2 results on '"Bell NM"'

1. Single-molecule DNA sequencing of widely varying GC-content using nucleotide release, capture and detection in microdroplets.

Author

Puchtler TJ, Johnson K, Palmer RN, Talbot EL, Ibbotson LA, Powalowska PK, Knox R, Shibahara A, M S Cunha P, Newell OJ, Wu M, Chana J, Athanasopoulou EN, Waeber AM, Stolarek M, Silva AL, Mordaka JM, Haggis-Powell M, Xyrafaki C, Bush J, Topkaya IS, Sosna M, Ingham RJ, Huckvale T, Negrea A, Breiner B, Šlikas J, Kelly DJ, Dunning AJ, Bell NM, Dethlefsen M, Love DM, Dear PH, Kuleshova J, Podd GJ, Isaac TH, Balmforth BW, and Frayling CA

Subjects
Base Composition genetics, Humans, Nanotechnology, Nucleotides genetics, DNA genetics, High-Throughput Nucleotide Sequencing, Sequence Analysis, DNA methods, Single Molecule Imaging
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., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Published
2020
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2. Single-molecule detection of deoxyribonucleoside triphosphates in microdroplets.

Author

Breiner B, Johnson K, Stolarek M, Silva AL, Negrea A, Bell NM, Isaac TH, Dethlefsen M, Chana J, Ibbotson LA, Palmer RN, Bush J, Dunning AJ, Love DM, Pachoumi O, Kelly DJ, Shibahara A, Wu M, Sosna M, Dear PH, Tolle F, Petrini E, Amasio M, Shelford LR, Saavedra MS, Sheridan E, Kuleshova J, Podd GJ, Balmforth BW, and Frayling CA

Subjects
DNA-Directed DNA Polymerase metabolism, Deoxyribonucleosides chemistry, Deoxyribonucleotides chemistry, Limit of Detection, Microscopy, Fluorescence, Oligodeoxyribonucleotides biosynthesis, Oligodeoxyribonucleotides chemistry, Sensitivity and Specificity, Deoxyribonucleotides analysis, High-Throughput Nucleotide Sequencing methods, Sequence Analysis, DNA methods
Abstract

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., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Published
2019
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