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A Droplet-Based Microfluidic Approach and Microsphere-PCR Amplification for Single-Stranded DNA Amplicons
Published on: November 14, 2018
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Digital PCR using a simple PDMS microfluidic chip and standard laboratory equipment
Kazuo Hosokawa1, Hitoshi Ohmori2
1Materials Fabrication Laboratory, RIKEN Cluster for Pioneering Research, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan. k-hoso@riken.jp.
Summary
This study introduces an affordable digital PCR (dPCR) system using a microfluidic chip and standard lab equipment. This innovation makes precise nucleic acid quantification accessible to more laboratories.
Area of Science:
- Molecular Biology
- Biotechnology
- Analytical Chemistry
Background:
- Digital PCR (dPCR) offers sensitive nucleic acid quantification without calibration.
- Widespread adoption of dPCR is limited by the high cost of specialized instrumentation.
Purpose of the Study:
- To develop an accessible and cost-effective dPCR system.
- To demonstrate the feasibility of using common laboratory tools for dPCR.
Main Methods:
- A microfluidic chip with 24,840 microwells was fabricated using PDMS and glass.
- PDMS degas-driven liquid control confined PCR mixtures to microwells.
- Standard thermal cyclers and a handmade water holder were used for thermal cycling and evaporation prevention.
Main Results:
- Clear discrimination between positive and negative microwells was achieved via fluorescence imaging.
- Template concentrations were accurately estimated using Poisson distribution theory.
- The system demonstrated accurate quantification across a wide concentration range (1.32–13,200 copies/µL).
Conclusions:
- The developed dPCR system provides a low-cost alternative to expensive commercial instruments.
- This approach facilitates broader implementation of digital PCR in diverse laboratory settings.
- The methodology enables sensitive and precise nucleic acid quantification without external calibration.
