Evaluating Droplet Digital PCR for the quantification of human genomic DNA: Lifting the Traceability Fog

Published

March 28, 2017

Author(s)

David L. Duewer, Margaret C. Kline

Abstract

Digital polymerase chain reaction (dPCR) endpoint platforms directly estimate the number of DNA target copies per reac-tion container, λ, where the containers are fixed-location chambers (cdPCR) or aqueous droplets floating in oil (ddPCR). For use in the certification of target concentration in primary calibrant certified reference materials (CRMs), both λ and the con-tainer volume, V, must be metrologically traceable to some accessible reference system: ideally, the International System of Units (SI). The fixed spatial distribution of cdPCR chambers enables real-time monitoring of PCR amplification. Analysis of the resulting reaction curves enables validation of the assumptions that are essential for establishing the SI-traceability of λ. We know of no direct method for validating these assumptions for ddPCR platforms. The manufacturers of the cdPCR and ddPCR systems available to us do not provide traceable container volume specifications. Colleagues at our institution have developed a reliable method for determining ddPCR droplet volume and have demonstrated that different ddPCR reagents yield droplets of somewhat different size. Thus neither dPCR platform by itself provides metrologically traceable estimates of target concentration. We show here that evaluating split-samples with both cdPCR and ddPCR platforms can transfer the λ traceability characteristics of a cdPCR assay to its ddPCR analogue, establishing fully traceable ddPCR esti-mates of CRM target concentration.

Citation

Analytical Chemistry

Volume

89

Issue

8

Pub Type

Journals

Download Paper

DOI Link

Keywords

Standard Reference Material, traceability, droplet digital Polymerase chain reaction

Citation

Duewer, D. and Kline, M. (2017), Evaluating Droplet Digital PCR for the quantification of human genomic DNA: Lifting the Traceability Fog, Analytical Chemistry, [online], https://doi.org/10.1021/acs.analchem.7b00240 (Accessed December 21, 2024)