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.

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

“…Four sets of oligonucleotides are used (one for each base-type and with each set labelled with a different fluorescent dye) in an enzymatic reaction to detect the presence and type of dNTP in a droplet. This dNTP detection process improves upon our previously published method ( 39 ), in terms of signal-to-noise ratio, and more importantly introduces the ability to tailor the reagents for detection of nucleotide modifications. This is done through the addition of a step involving a restriction enzyme which, if chosen to be modification-sensitive, opens the door to modification detection.…”

“…We observed a dNTP capture efficiency close to 100% (Supplementary Information, Supplementary Figure S4 ) in our preliminary analysis performed in bulk solution. Additionally, the dNTP capture specificity using Bst L.F. in this type of reaction is >99.9% ( 39 ). The efficacy of the PPL reaction was also confirmed under bulk solution conditions (Supplementary Information, Supplementary Figure S5 ).…”

“…The sequencing workflow requires precise manipulation of ∼100-femtoliter-scale microdroplets to control dNTP release and addition of detection reagents. Droplets of this scale are required to ensure that the presence of a single dNTP within a droplet results in a sufficiently high concentration of fluorophores to generate an adequate signal-to-noise ratio in the dNTP detection reaction (see Supplementary Information, Supplementary Figure S3 and ( 39 )). In addition, smaller droplets offer greater scalability as they result in less device area per base sequenced.…”

A sensitive assay for dNTPs based on long synthetic oligonucleotides, EvaGreen dye and inhibitor-resistant high-fidelity DNA polymerase

“…Recent findings have also questioned the robustness of the probe-based detection with biological samples ( 26 ). Recently, a rather elaborate enzymatic two-stage dNTP detection involving a fluorescent probe, two DNA fragments, two different DNA polymerases and a DNA ligase was developed ( 27 ). This assay could reach a detection limit that is orders of magnitude lower than any other existing method could.…”

A sensitive assay for dNTPs based on long synthetic oligonucleotides, EvaGreen dye, and inhibitor-resistant high-fidelity DNA polymerase

“…Less stringent criteria (5 SD over background) for LLOQ was, however, used by these authors than by us. Recently, a rather elaborate enzymatic two-stage dNTP detection involving a fluorescent probe, two DNA fragments, two different DNA polymerases and a DNA ligase was developed (14). This assay could reach a detection limit that is orders of magnitude lower than any other existing method could.…”