Anal Bioanal Chem DOI 10.1007/s00216-016-0131-7 RESEARCH PAPER Clarity™ digital PCR system: a novel platform for absolute quantification of nucleic acids Huiyu Low 1 & Shun-Jie Chan 2 & Guo-Hao Soo 1 & Belinda Ling 1 & Eng-Lee Tan 2,3 Received: 27 September 2016 / Revised: 17 November 2016 / Accepted: 2 December 2016 # Springer-Verlag Berlin Heidelberg 2016 Abstract In recent years, digital polymerase chain reaction (dPCR) has gained recognition in biomedical research as it provides a platform for precise and accurate quantification of nucleic acids without the need for a standard curve. However, this technology has not yet been widely adopted as compared to real-time quantitative PCR due to its more cumbersome workflow arising from the need to sub-divide a PCR sample into a large number of smaller partitions prior to thermal cycling to achieve zero or at least one copy of the target RNA/ DNA per partition. A recently launched platform, the Clarity™ system from JN Medsys, simplifies dPCR workflow through the use of a novel chip-in-a-tube technology for sample partitioning. In this study, the performance of Clarity™ was evaluated through quantification of the single-copy human RNase P gene. The system demonstrated high precision and accuracy and also excellent linearity across a range of over 4 orders of magnitude for the absolute quantification of the target gene. Moreover, consistent DNA copy measurements were also attained using a panel of different probeand dye-based master mixes, demonstrating the system’s compatibility with commercial master mixes. The Clarity™ was Electronic supplementary material The online version of this article (doi:10.1007/s00216-016-0131-7) contains supplementary material, which is available to authorized users. * Eng-Lee Tan [email protected] 1 JN Medsys, 217 Henderson Road #02-08, Singapore 159555, Singapore Centre for Biomedical and Life Sciences, Singapore Polytechnic, 500 Dover Road, Singapore 139651, Singapore Department of Paediatrics, University Children’s Medical Institute, National University Hospital, 1E Kent Ridge Road, Singapore 119228, Singapore then compared to the QX100™ droplet dPCR system from Bio-Rad using a set of DNA reference materials, and the copy number concentrations derived from both systems were found to be closely associated. Collectively, the results showed that Clarity™ is a reliable, robust and flexible platform for nextgeneration genetic analysis. Keywords Tube-strip digital PCR . Absolute DNA quantification . Sample partitioning . Clarity Introduction Digital polymerase chain reaction (dPCR) is the thirdgeneration PCR technology that enables absolute quantification of target nucleic acid without using external standards. It works by dividing a PCR mixture into thousands of independent nanolitre partitions, such that each individual reaction contains zero or at least one target of interest [1]. By analysing the proportion of partitions that are positive or negative for PCR products, an absolute DNA copy number is then determined based on the Poisson statistics. Apart from obviating the use of a DNA standard, dPCR has additional advantages over real-time quantitative PCR (qPCR) such as improved precision and accuracy, lower susceptibility to PCR inhibitors and increased sensitivity for detecting rare targets amidst a high background of non-target DNA [1]. Due to its advantages, dPCR has successfully been applied to a wide array of preclinical research studies such as rare mutation detection, copy number variation studies and low-level pathogen quantification [2–6]. Commercial dPCR platforms mainly employ two different methods of sample partitioning. The QX100™/QX200™ (Bio-Rad Laboratories) and RainDrop ® (RainDance™ Technologies) systems partiti

formation of water-in-oil droplets while the BioMark™ HD (Fluidigm) and QuantStudio® 3D (Thermo Fisher Scientific) systems employ chip-based technology to generate partitions. Droplet-based platforms typically have wider dynamic ranges of detection as they generate a higher number of partitions per sample. Their use of conventional multi-well PCR plates also reduces the complexity of performing dPCR. Droplets, however, may coalesce during thermal cycling [7, 8], and this results in sample loss which is a major drawback for analysis of samples with low abundance DNA. On the other hand, chip-based...

Absolute nucleic acid quantification using Clarity™ dPCR Table 1 Oligonucleotides used for BCR-ABL b3a2 amplification BCR-ABL b3a2 forward BCR-ABL b3a2 reverse BCR-ABL b3a2 probe proprietary formulation optimized for robust performance on Clarity™ high-density chips. Using the Clarity™ auto loader, the resultant mix was then delivered onto the chip where it was sub-divided into 10,000 partitions. The partitions were then sealed with the Clarity™ Sealing Enhancer and 230 μL Clarity™ Sealing Fluid, followed by thermal cycling using the following parameters: initial cycle of 95 °C for 10 min and...

Measured Concentraion (copies/ L) 10 100 1000 Expected Concentration (copies/ L) Fig. 2 Measured concentrations of RNase P using Clarity™ against its expected values. The graph is represented in a log-log format with each data point representing the mean ± standard deviation of five replicates spectrophotometry). The prepared master mixes were then subjected to Clarity™ dPCR analysis using experimental procedures as described in the BQuantification of the human RNase P gene using the Clarity™ dPCR system^ section. Quantification of ERM-AD623 by Clarity™ and QX100™ The ERM-AD623 reference materials...

Absolute nucleic acid quantification using Clarity™ dPCR Results and discussion Clarity™ dPCR system quantifies DNA with high accuracy and precision As Clarity™ is a newly launched dPCR system, the current study was initiated to evaluate its accuracy and precision in the absolute quantification of target DNA. To achieve this, eight serial dilutions of the purified human genomic DNA were prepared and used as templates for dPCR amplification of RNase P. In this experiment, the mean number of partitions detected was 9782 with a standard deviation of 142 partitions (see Electronic Supplementary Material...