Dataset Information

Single-molecule detection of deoxyribonucleoside triphosphates in microdroplets.

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.

SUBMITTER: Breiner B

PROVIDER: S-EPMC6753480 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Publications

Single-molecule detection of deoxyribonucleoside triphosphates in microdroplets.

Breiner Boris B Johnson Kerr K Stolarek Magdalena M Silva Ana-Luisa AL Negrea Aurel A Bell Neil M NM Isaac Tom H TH Dethlefsen Mark M Chana Jasmin J Ibbotson Lindsey A LA Palmer Rebecca N RN Bush James J Dunning Alexander J AJ Love David M DM Pachoumi Olympia O Kelly Douglas J DJ Shibahara Aya A Wu Mei M Sosna Maciej M Dear Paul H PH Tolle Fabian F Petrini Edoardo E Amasio Michele M Shelford Leigh R LR Saavedra Monica S MS Sheridan Eoin E Kuleshova Jekaterina J Podd Gareth J GJ Balmforth Barnaby W BW Frayling Cameron A CA

Nucleic acids research 20190901 17

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 ...[more]

PMID: 31318971

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Project description:Over the past few decades, single molecule investigations employing optical tweezers, AFM and TIRF microscopy have revealed that molecular behaviors are typically characterized by discrete steps or events that follow changes in protein conformation. These events, that manifest as steps or jumps, are short-lived transitions between otherwise more stable molecular states. A major limiting factor in determining the size and timing of the steps is the noise introduced by the measurement system. To address this impediment to the analysis of single molecule behaviors, step detection algorithms incorporate large records of data and provide objective analysis. However, existing algorithms are mostly based on heuristics that are not reliable and lack objectivity. Most of these step detection methods require the user to supply parameters that inform the search for steps. They work well, only when the signal to noise ratio (SNR) is high and stepping speed is low. In this report, we have developed a novel step detection method that performs an objective analysis on the data without input parameters, and based only on the noise statistics. The noise levels and characteristics can be estimated from the data providing reliable results for much smaller SNR and higher stepping speeds. An iterative learning process drives the optimization of step-size distributions for data that has unimodal step-size distribution, and produces extremely low false positive outcomes and high accuracy in finding true steps. Our novel methodology, also uniquely incorporates compensation for the smoothing affects of probe dynamics. A mechanical measurement probe typically takes a finite time to respond to step changes, and when steps occur faster than the probe response time, the sharp step transitions are smoothed out and can obscure the step events. To address probe dynamics we accept a model for the dynamic behavior of the probe and invert it to reveal the steps. No other existing method addresses the impact of probe dynamics on step detection. Importantly, we have also developed a comprehensive set of tools to evaluate various existing step detection techniques. We quantify the performance and limitations of various step detection methods using novel evaluation scales. We show that under these scales, our method provides much better overall performance. The method is validated on different simulated test cases, as well as experimental data.

Dataset Information

Single-molecule detection of deoxyribonucleoside triphosphates in microdroplets.

Publications

Single-molecule detection of deoxyribonucleoside triphosphates in microdroplets.

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