Methods for Multiplex Template Sampling in Digital PCR Assays

Abstract

The efficient use of digital PCR (dPCR) for precision copy number analysis requires high concentrations of target molecules that may be difficult or impossible to obtain from clinical samples. To solve this problem we present a strategy, called Multiplex Template Sampling (MTS), that effectively increases template concentrations by detecting multiple regions of fragmented target molecules. Three alternative assay approaches are presented for implementing MTS analysis of chromosome 21, providing a 10-fold concentration enhancement while preserving assay precision.

Use of ddPCR in experimental evolution studies

“…; Petriv et al . ), also make it ideal for basic research and EES. Performing a multiplex assay allows the detection of two target molecules in a sample by using different fluorescent dyes (Channel 1: FAM ™ ; Channel 2: HEX ™ /VIC ® ).…”

“…Pohl & Shih 2004). Its accuracy and ability to detect small differences in target concentration, coupled with multiplexing capabilities (Zhong et al 2011;McDermott et al 2013;Petriv et al 2014), also make it ideal for basic research and EES. Performing a multiplex assay allows the detection of two target molecules in a sample by using different fluorescent dyes (Channel 1: FAM TM ; Channel 2: HEX TM / VIC Ò ).…”

Segmental duplication as potential biomarkers for non-invasive prenatal testing of aneuploidies

“…The uniqueness of SD sequences-based PCR assays lies in the use of a common pair of primers to amplify a pair of SD sequences located in the respective target and the reference chromosomes. Thus, virtually identical amplification efficiency for both sequences can be achieved even when multiple primers are present or even when the reaction conditions fluctuate [21]. This is critical in the particular case of aneuploidy detection, where a small amplification bias might mitigate the small copy number differences between euploidy and aneuploidy.…”

From single-molecule detection to next-generation sequencing: microfluidic droplets for high-throughput nucleic acid analysis

“…Accordingly, in multiplex ddPCR, multiple mutations can be detected in a single experiment; a feature particularly valuable when assaying clinical samples (Taly et al 2013). Much work has recently focused on improving ddPCR, in terms of detection sensitivity (Miotke et al 2014) and sample volume limitation (Petriv et al 2014), but there is little doubt that ddPCR is rapidly becoming a "standard" component in highly sensitive genomic screening.…”