Next generation multiplexing for digital PCR using a novel melt-based hairpin probe design

Abstract

Digital PCR (dPCR) is a powerful tool for research and diagnostic applications that require absolute quantification of target molecules or detection of rare events, but the number of nucleic acid targets that can be distinguished within an assay has limited its usefulness. For most dPCR systems, one target is detected per optical channel and the total number of targets is limited by the number of optical channels on the platform. Higher-order multiplexing has the potential to dramatically increase the usefulness of dPCR, especially in scenarios with limited sample. Other potential benefits of multiplexing include lower cost, additional information generated by more probes, and higher throughput. To address this unmet need, we developed a novel melt-based hairpin probe design to provide a robust option for multiplexing digital PCR. A prototype multiplex digital PCR (mdPCR) assay using three melt-based hairpin probes per optical channel in a 16-well microfluidic digital PCR platform accurately distinguished and quantified 12 nucleic acid targets per well. For samples with 10,000 human genome equivalents, the probe-specific ranges for limit of blank were 0.00%-0.13%, and those for analytical limit of detection were 0.00%-0.20%. Inter-laboratory reproducibility was excellent (r ² = 0.997). Importantly, this novel melt-based hairpin probe design has potential to achieve multiplexing beyond the 12 targets/well of this prototype assay. This easy-to-use mdPCR technology with excellent performance characteristics has the potential to revolutionize the use of digital PCR in research and diagnostic settings.

Keywords: absolute quantification; digital PCR; higher-order multiplexing; mdPCR assay; melt-based hairpin probe design.

FIGURE 1

Key aspects of mdPCR. (A) Melt-based probe design. (B) The probe binds to its amplicon target, causing the formation of an RNA:DNA duplex at the ribobase (r) present in the probe. RNAse H2 present in the reaction mixture cleaves the probe, allowing the 5′ portion of the cleaved probe to form a hairpin which is extended by DNA polymerase. During extension, the DNA polymerase directs a site-specific incorporation of a fluorescent quencher to form a temperature-reversible quenched hairpin. (C) Post-amplification, probes containing the same fluorophore can be discriminated based on the hybridization temperature of the final hairpin as determined by discrete melt analysis in which the fluorescence is measured by imaging at four temperatures and the ratios of each set of temperatures (T2/T1, T3/T2, and T4/T3) are used to determine presence/absence of each target. (D) Workflow for Erebos data analysis. Optimized positivity thresholds (ratios) for each probe (previously determined experimentally) used were: P1-A > 1.075, P1-B > 1.1, P2-A > 1.06, P2-B > 1.11, P3-A > 1.08, P3-B > 1.12, P4-A > 1.07, P4-B > 1.1, P5-A > 1.085, P5-B > 1.1, P6-A > 1.08 and P6-B 1.075. The copies per μL, total copies per assay input, allele frequency, and 95% Poisson confidence intervals were calculated for each target.

FIGURE 2

mdPCR assay results. (A) Representative data plots and tables from reactions containing 10,000, 5,000, and 1,000 human genomic equivalents from a single individual. Within each partition, 12 targets were simultaneously detected, and Poisson statistics were used to quantify each target in each reaction. For each locus (P1—P6) there were two alleles which were designated (A, B). Partitions below a pre-determined positive RFU ratio were considered to have no target and are shown as gray points which are near 1.0. Partitions above a pre-determined positive RFU ratio were defined as positive and have a different color for each channel (green, red, yellow, blue). (B) mdPCR assay results for 12 targets, minor fraction 0.3%–50%. The graphs (on log10 scales) show the number of copies for each minor allele that was detected for each probe (P1-A—P6-B). The results ranged from 30 to 5,000 copies for each of the four reactions. These experiments were carried out using mixtures of two human genomic DNA samples containing minor fractions of each allele at 0%, 0.3%, 0.5%, 1%, 2%, 5%, 10%, 25% or 50%. Four replicates of each sample were tested. DNA input was 10, 000 hge (33.3 ng of DNA). Error bars represent the 95% confidence interval. The CVs for these measurements are provided in Supplementary Datasheet S2.

FIGURE 3

Inter-laboratory reproducibility. The graph shows absolute values of DNA minor fraction concentrations ranging from 30 to 5,000 copies per reaction (n = 4) measured at site 1 and site 2. Data are shown on log10 scales. Correlation between site 1 and site 2 is r = 0.997.