title: Fundamentals of multiplexing with digital PCR.
abstract: Over the past decade numerous publications have demonstrated how digital PCR (dPCR) enables precise and sensitive quantification of nucleic acids in a wide range of applications in both healthcare and environmental analysis. This has occurred in parallel with the advances in partitioning fluidics that enable a reaction to be subdivided into an increasing number of partitions. As the majority of dPCR systems are based on detection in two discrete optical channels, most research to date has focused on quantification of one or two targets within a single reaction. Here we describe 'higher order multiplexing' that is the unique ability of dPCR to precisely measure more than two targets in the same reaction. Using examples, we describe the different types of duplex and multiplex reactions that can be achieved. We also describe essential experimental considerations to ensure accurate quantification of multiple targets.
authors.affiliation: Molecular and Cell Biology Team, LGC, Queens Road, Teddington, Middlesex TW11 0LY, United Kingdom.
authors.forename: Alexandra S
authors.initials: AS
authors.lastname: Whale
authors.affiliation: Molecular and Cell Biology Team, LGC, Queens Road, Teddington, Middlesex TW11 0LY, United Kingdom.
authors.forename: Jim F
authors.initials: JF
authors.lastname: Huggett
authors.affiliation: Digital Biology Centre, Bio-Rad Laboratories Inc., 5731 West Las Positas Boulevard, Pleasanton, CA 94588, United States.
authors.forename: Svilen
authors.initials: S
authors.lastname: Tzonev
keywords: Digital PCR; Duplex; Higher order multiplexing; Multiplexing; dPCR