Base4 Innovation Ltd, Broers Building, 21 JJ Thomson Avenue, Cambridge CB3 0FA, UK.
Nucleic Acids Res. 2019 Sep 26;47(17):e101. doi: 10.1093/nar/gkz611.
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.
一种新的单分子 DNA 测序方法，其中待测序链上的焦磷酸解释放的 dNTP 被微滴捕获并直接读取，与当前的序列合成方法相比可能具有显著优势；然而，目前还没有足够灵敏的方法可以在微滴中检测单个核苷酸。我们开发了一种基于酶的两阶段反应的 dNTP 检测方法，该方法产生了一种稳健的荧光信号，易于检测和处理。利用 DNA 聚合酶和连接酶的固有特异性，结合微滴中的体积限制，这种方法使我们能够在单分子水平上同时检测和区分四种天然 dNTP 的存在，几乎没有串扰。
