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Two-phase wash to solve the ubiquitous contaminant-carryover problem in commercial nucleic-acid extraction kits

Jue, Erik and Witters, Daan and Ismagilov, Rustem F. (2020) Two-phase wash to solve the ubiquitous contaminant-carryover problem in commercial nucleic-acid extraction kits. Scientific Reports, 10 . Art. No. 1940. ISSN 2045-2322. PMCID PMC7004994. doi:10.1038/s41598-020-58586-3.

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The success of fundamental and applied nucleic acid (NA) research depends on NA purity, but obtaining pure NAs from raw, unprocessed samples is challenging. Purification using solid-phase NA extractions utilizes sequential additions of lysis and wash buffers followed by elution. The resulting eluent contains NAs and carryover of extraction buffers. Typically, these inhibitory buffers are heavily diluted by the reaction mix (e.g., 10x dilution is 1 µL eluent in 9 µL reaction mix), but in applications requiring high sensitivity (e.g., single-cell sequencing, pathogen diagnostics) it is desirable to use low dilutions (e.g., 2x) to maximize NA concentration. Here, we demonstrate pervasive carryover of inhibitory buffers into eluent when several commercial sample-preparation kits are used following manufacturer protocols. At low eluent dilution (2–2.5x) we observed significant reaction inhibition of polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), and reverse transcription (RT). We developed a two-phase wash (TPW) method by adding a wash buffer with low water solubility prior to the elution step. The TPW reduces carryover of extraction buffers, phase-separates from the eluent, and does not reduce NA yield (measured by digital PCR). We validated the TPW for silica columns and magnetic beads by demonstrating significant improvements in performance and reproducibility of qPCR, LAMP, and RT reactions.

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URLURL TypeDescription ItemData CentralArticle
Jue, Erik0000-0001-7585-3794
Witters, Daan0000-0003-2179-5300
Ismagilov, Rustem F.0000-0002-3680-4399
Additional Information:© The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit Received 24 October 2019. Accepted 2 January 2020. Published 6 February 2020. This work was supported in part by the Defense Threat Reduction Agency (DTRA) award MCDC-18-01-01-007, an effort sponsored by the U.S. Government under Other Transaction number W15QKN-16-9-1002 between the MCDC, and the Government. The U.S. Government is authorized to reproduce and distribute reprints for Governmental purposes notwithstanding any copyright notation thereon. The views and conclusions contained herein are those of the authors and should not be interpreted as necessarily representing the official policies or endorsements, either expressed or implied, of the U.S. Government. This work was also supported by a Burroughs Wellcome Fund Innovation in Regulatory Science Award, a National Science Foundation Graduate Research Fellowship DGE-1144469 (to E.J.), an NIH National Research Service Award (NRSA) [5T32GM07616NSF] (to E.J.), a grant from the Rothenberg Innovation Initiative (RI2) and a grant from the Joseph J. Jacobs Institute for Molecular Engineering for Medicine. We thank Kevin Winzey for running the buffer-dilution experiments for Figure 2, Nathan Schoepp for optimized reaction conditions for LAMP, E. coli extractions and E. coli primers, Pedro Ojeda for running exploratory experiments with ethanol and octanol spiked into LAMP, Andrew Friedman for running exploratory experiments with octanol and 1-undecanol TPW extractions, and Natasha Shelby for contributions to writing and editing this manuscript. Author Contributions: E.J. Acquired funding. Planned and analyzed buffer dilution experiments for Figure 2. Planned, ran, and analyzed all remaining experiments. Generated all figures, and wrote the manuscript. D.W. Ran preliminary experiments evaluating the use of TPW to reduce buffer carryover. R.F.I. Supervised the project, acquired funding, helped to analyze the data, and reviewed and edited the manuscript. Full dataset available through CaltechDATA,; Competing interests: The content of this manuscript is the subject of a patent application filed by Caltech. R.F.I. has a financial interest in Talis Biomedical Corp.; all other authors have no conflict of interest.
Group:Jacobs Institute for Molecular Engineering for Medicine
Funding AgencyGrant Number
Defense Threat Reduction Agency (DTRA)MCDC-18-01-01-007
Defense Threat Reduction Agency (DTRA)W15QKN-16-9-1002
Burroughs Wellcome FundUNSPECIFIED
NIH Predoctoral Fellowship5T32GM07616NSF
Rothenberg Innovation Initiative (RI2)UNSPECIFIED
NSF Graduate Research FellowshipDGE-1144469
Jacobs Institute for Molecular Engineering for MedicineUNSPECIFIED
PubMed Central ID:PMC7004994
Record Number:CaltechAUTHORS:20200206-125057522
Persistent URL:
Official Citation:Jue, E., Witters, D. & Ismagilov, R.F. Two-phase wash to solve the ubiquitous contaminant-carryover problem in commercial nucleic-acid extraction kits. Sci Rep 10, 1940 (2020).
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:101169
Deposited By: George Porter
Deposited On:07 Feb 2020 18:33
Last Modified:16 Nov 2021 18:00

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