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Digital PCR modeling for maximal sensitivity, dynamic range and measurement precision.

Authors :
Nivedita Majumdar
Thomas Wessel
Jeffrey Marks
Source :
PLoS ONE, Vol 10, Iss 3, p e0118833 (2015)
Publication Year :
2015
Publisher :
Public Library of Science (PLoS), 2015.

Abstract

The great promise of digital PCR is the potential for unparalleled precision enabling accurate measurements for genetic quantification. A challenge associated with digital PCR experiments, when testing unknown samples, is to perform experiments at dilutions allowing the detection of one or more targets of interest at a desired level of precision. While theory states that optimal precision (Po) is achieved by targeting ~1.59 mean copies per partition (λ), and that dynamic range (R) includes the space spanning one positive (λL) to one negative (λU) result from the total number of partitions (n), these results are tempered for the practitioner seeking to construct digital PCR experiments in the laboratory. A mathematical framework is presented elucidating the relationships between precision, dynamic range, number of partitions, interrogated volume, and sensitivity in digital PCR. The impact that false reaction calls and volumetric variation have on sensitivity and precision is next considered. The resultant effects on sensitivity and precision are established via Monte Carlo simulations reflecting the real-world likelihood of encountering such scenarios in the laboratory. The simulations provide insight to the practitioner on how to adapt experimental loading concentrations to counteract any one of these conditions. The framework is augmented with a method of extending the dynamic range of digital PCR, with and without increasing n, via the use of dilutions. An example experiment demonstrating the capabilities of the framework is presented enabling detection across 3.33 logs of starting copy concentration.

Subjects

Subjects :
Medicine
Science

Details

Language :
English
ISSN :
19326203
Volume :
10
Issue :
3
Database :
Directory of Open Access Journals
Journal :
PLoS ONE
Publication Type :
Academic Journal
Accession number :
edsdoj.7fa8a22b72540eb83ee471e2f154b62
Document Type :
article
Full Text :
https://doi.org/10.1371/journal.pone.0118833