Sub-nanoliter Multiple Displacement Amplification of DNA Using Droplet-based Microfluidic Systems

M. Rhee, R.J. Meagher, Y.K. Light, A.K. Singh
Sandia National Laboratories, US

Keywords: multiple displacement amplification, droplet microfluidics

Summary:

We present here a droplet-based microfluidic system for amplifying bacterial genomic DNA in sub-nanoliter droplets by isothermal multiple displacement amplification (MDA). We integrated an on-demand droplet generation module with a picoinjecting module on a single chip to process multiple steps during the course of droplet transportation. DNA-containing droplets were first generated and MDA reagent mix including polymerase ϕ29 enzyme was injected into the flowing droplets thereafter, which enabled to avoid premature amplification or contamination. We also demonstrated the integrated chip for on-demand encapsulation of cells followed by cell lysis and DNA denaturization within droplets by injection of alkaline lysis reagents. Our unique method for sub-nanoliter volume MDA is particularly appropriate for high throughput screening, enzymatic assays and uncultivated cell studies, and genomic assays as well as precise quantification of DNA for forensics, water quality control, and counter-bioterrorism. This method will be also well suited for metagenomic studies to analyze extremely dilute samples such as pathogens in a clinical sample.