A droplet-based microfluidic platform for kinetics-based detection of single nucleotide variation at room temperature with large discrimination factors

Abstract

In this paper we presented a droplet-based microfluidic platform for the detection of single nucleotide variation by measuring the kinetics of Klenow Fragments (exo-) based extension reaction. In the platform, nanoliter droplets containing the DNA sequence, Klenow Fragments (exo-), buffer and dNTPs or rNTPs mixture were formed in the microchannel, which acted as microreactors. Target DNA sequences with and without single nucleotide variation were used as primers during the extension. For the rapid kinetics with matching template-primer duplex, the reaction time was directly related to the droplets’ traveling distance. For the slow kinetics with mismatching template-primer duplex, the flow was stopped after the formation of the droplets, and then the selected droplets were monitored to measure the kinetics. The droplet-based platform presented a dead time of sub-seconds and a wide range of observation time from sub-seconds to hundreds of minutes. The platform allowed the measurement of reaction kinetics ranging from s−1 to 10−4s−1 and achieved large discrimination factors ranging from 100 to 550 for the detection of single nucleotide variation.