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Single-molecule detection of deoxyribonucleoside triphosphates in microdroplets. 2019
Boris Breiner, and
Kerr Johnson, and
Magdalena Stolarek, and
Ana-Luisa Silva, and
Aurel Negrea, and
Neil M Bell, and
Tom H Isaac, and
Mark Dethlefsen, and
Jasmin Chana, and
Lindsey A Ibbotson, and
Rebecca N Palmer, and
James Bush, and
Alexander J Dunning, and
David M Love, and
Olympia Pachoumi, and
Douglas J Kelly, and
Aya Shibahara, and
Mei Wu, and
Maciej Sosna, and
Paul H Dear, and
Fabian Tolle, and
Edoardo Petrini, and
Michele Amasio, and
Leigh R Shelford, and
Monica S Saavedra, and
Eoin Sheridan, and
Jekaterina Kuleshova, and
Gareth J Podd, and
Barnaby W Balmforth, and
Cameron A Frayling
Base4 Innovation Ltd, Broers Building, 21Â JJ Thomson Avenue, Cambridge CB3 0FA, UK.
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.
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