Evaluating Digital PCR as a primary method for the quantification of human genomic DNA: Accessible amplifiable targets
Margaret C. Kline, Erica L. Romsos, David L. Duewer
Polymerase chain reaction (PCR) assays perform best when the input quantity of template DNA is controlled to within about a factor of √2. To help ensure that PCR assays yield consistent results over time and place, results from methods used to determine DNA quantity need to be metrologically traceable to a common reference. Many DNA quantitation sys-tems can be accurately calibrated with solutions of DNA in aqueous buffer. Since they do not require external calibration, end-point limiting dilution technologies, collectively termed "digital PCR (dPCR)", have been proposed as suitable as a pri-mary method for value assigning such DNA calibrants. The performance characteristics of several commercially available dPCR systems have recently been documented using plasmid, viral, or fragmented genomic DNA; dPCR performance with more complex materials, such as human genomic DNA, has been less studied. With the goal of providing a human genomic reference material traceably certified for mass concentration, we are investigating the measurement characteristics of sever-al dPCR systems. We here report results of measurements from four PCR assays, on four human genomic DNA extracts from different sources subjected to four different endonuclease restriction enzyme treatments using a dPCR system that tracks the progress of PCR amplification in multiple reaction chambers. We conclude that dPCR does not estimate the ab-solute number of PCR targets in a given volume but rather the number of accessible and amplifiable targets. Results from several well-characterized PCR assays, targeting different chromosomes, are needed to adequately characterize the biases and variability of dPCR assays.