Digital polymerase chain reaction measured pUC19 marker as calibrant for HPLC measurement of DNA quantity.

ABSTRACT

Digital polymerase chain reaction (dPCR) is potentially a primary method for quantifying target DNA regions in a background of nontarget material and is independent of external calibrators. Accurate dPCR measurements require single-molecule detection by conventional PCR assays that may be subject to bias due to inhibition, interference, or sequence-derived PCR inefficiency. Elimination or control of such biases is essential for validation of PCR assays, but this may require a substantial investment in resources. Here we present a mechanism for DNA quantification that does not require PCR assay validation in situations where target DNA quantity is high enough to be measured by physical techniques such as quantitative high-performance liquid chromatography (HPLC) or electrophoresis. A commercially available DNA marker derived from pUC19 was quantified by dPCR and was then used to calibrate an HPLC measuring system for quantifying a DNA amplicon that had a high content of guanidine and cytidine. The dPCR-calibrated HPLC measurement was verified by independent measurement using isotope dilution mass spectrometry (IDMS). HPLC quantification, calibrated with dPCR or IDMS measured DNA markers, provides an effective method for certifying the quantity of genetic reference materials that may be difficult to analyze by PCR. These secondary reference materials may then be used to validate and calibrate quantitative PCR measurements and thus could expand the breadth of applications for which traceability to the International System of Units is possible.