Droplet Digital Polymerase Chain Reaction: A Method to Generate Nanodroplet PCR Reactions for Detecting Rare Tumor Mutations

Abstract

Source: Bonner, E. R. et al. Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids. J. Vis. Exp. (2019)

In this video, we present a step-by-step procedure to generate emulsified nano-droplets to detect target nucleic acids by digital polymerase chain reaction or dPCR.

Protocol

1. Detection and Quantification of Target DNA Using dPCR Prepare the dPCR reaction mixture for 8 wells of the PCR strip tube plus 10% extra, by adding 220 µL of genotyping master mix, 8.8 µL each of 10 µM wildtype and mutant probes, 39.6 µL each of 10 µM forward and reverse primers, and 17.6 µL of droplet stabilizing oil. Gently mix the dPCR reaction mixture by pipetting the full volume up and down 10–20 times. Do not vortex or centrifuge after dr…

Materials

Raindance Consumable Kit	Bio-Rad	20-04411	Contains droplet-generating instrument chips, quantification instrument chips, PCR strip caps including high-speed caps, and droplet stabilizing oil
Raindance Sense Instrument	Bio-Rad		Quantification instrument
Raindance Source Instrument	Bio-Rad		Droplet-generating instrument
RainDrop Analyst II Software	RainDance Technologies		Analyst software used for Data Analysis
Thermomixer C	Eppendorf	14 285 562PM
MiniAmp Thermal Cycler	Thermofisher Scientific	A37834
PCR 8 well strip tube caps	VWR	10011-786
PCR 8 well strip tubes	Axygen	PCR-0208-C
Q5 Hot Start High-Fidelity 2x Master Mix	New England Biolabs Inc	M0494S