Simple Bulk Readout of Digital Nucleic Acid Quantification Assays
Name
Morinishi-2015-Simple Bulk Readout.pdf
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517.43 KB
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Author(s)
Morinishi, Leanna S.
Blainey, Paul C.
Date Issued
September 2015
Journal
Journal of Visualized Experiments
Publisher
MyJoVE Corporation
Citation
Morinishi, Leanna S., and Paul Blainey. “Simple Bulk Readout of Digital Nucleic Acid Quantification Assays.” JoVE no. 103 (2015). © 2015 Journal of Visualized Experiments
Version
Final published version
Abstract
Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are still not routinely applied, due to the cost and specific equipment associated with commercially available methods. Here we present a simplified method for readout of digital droplet assays using a conventional real-time PCR instrument to measure bulk fluorescence of droplet-based digital assays.
We characterize the performance of the bulk readout assay using synthetic droplet mixtures and a droplet digital multiple displacement amplification (MDA) assay. Quantitative MDA particularly benefits from a digital reaction format, but our new method applies to any digital assay. For established digital assay protocols such as digital PCR, this method serves to speed up and simplify assay readout.
Our bulk readout methodology brings the advantages of partitioned assays without the need for specialized readout instrumentation. The principal limitations of the bulk readout methodology are reduced dynamic range compared with droplet-counting platforms and the need for a standard sample, although the requirements for this standard are less demanding than for a conventional real-time experiment. Quantitative whole genome amplification (WGA) is used to test for contaminants in WGA reactions and is the most sensitive way to detect the presence of DNA fragments with unknown sequences, giving the method great promise in diverse application areas including pharmaceutical quality control and astrobiology.
MIT Department
Massachusetts Institute of Technology. Department of Biological Engineering
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Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use.
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DOI of Published Version
https://doi.org/10.3791/52925
