Digital PCR using a simple PDMS microfluidic chip and standard laboratory equipment

Abstract

Digital PCR (dPCR) enables sensitive and precise quantification of template nucleic acid without calibration. However, dPCR is not yet in widespread use, probably due to the need for expensive specialized instruments. In this paper, we describe a dPCR system using a simple microfluidic chip and common laboratory tools. The microfluidic chip consists of two parts: a PDMS part with 24,840 × 0.25 nL microwells and a PDMS-coated flat glass plate. Human RNase P gene was adopted as the model template. Commercial products of human genomic DNA and real-time PCR reagents were mixed to make a PCR mixture. The PCR mixture was confined to the microwells by the PDMS degas-driven liquid control technique. The thermal cycling was performed on a common well-type thermal cycler with a minor modification. During the thermal cycling, evaporation of the PCR mixture was prevented with a handmade water holder. In the fluorescence image, bright (positive) microwells and dim (negative) ones were clearly discriminated. The number of the positive microwells was counted using software, and was used for estimation of the template concentration in the sample based on the theory of the Poisson distribution. The estimated concentrations well agreed with the input template concentrations in the range from 1.32 copies/µL to 13 200 copies/µL. The techniques presented in this paper will pave the way for facile dPCR in a broad range of laboratories without the need for expensive instruments.

Keywords: Degas-driven; Digital PCR; Microfluidic chip; Microwell; PDMS; Power-free.