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Micromachines 2018, 9(8), 367; https://doi.org/10.3390/mi9080367

The Development of an Effective Bacterial Single-Cell Lysis Method Suitable for Whole Genome Amplification in Microfluidic Platforms

1
Department of Surgery, Division of Surgical Research, Mayo Clinic, Rochester, MN 55905, USA
2
Astrobiology Group, Center of Astronomy and Astrophysics, Technical University, 10623 Berlin, Germany
3
German Aerospace Center (DLR), Institute of Planetary Research, Management and Infrastructure, Astrobiological Laboratories, 12489 Berlin, Germany
4
School of Physics and Astronomy, University of Edinburgh, Edinburgh EH9 3FD, UK
5
Fraunhofer Institute for Cell Therapy and Immunology, Branch Bioanalytics and Bioprocesses (IZI-BB), Extremophile Research & Biobank CCCryo, 14476 Potsdam, Germany
6
Department of Obstetrics and Gynecology, Mayo Clinic, Rochester, MN 55905, USA
*
Author to whom correspondence should be addressed.
Received: 7 June 2018 / Revised: 13 July 2018 / Accepted: 19 July 2018 / Published: 25 July 2018
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Abstract

Single-cell sequencing is a powerful technology that provides the capability of analyzing a single cell within a population. This technology is mostly coupled with microfluidic systems for controlled cell manipulation and precise fluid handling to shed light on the genomes of a wide range of cells. So far, single-cell sequencing has been focused mostly on human cells due to the ease of lysing the cells for genome amplification. The major challenges that bacterial species pose to genome amplification from single cells include the rigid bacterial cell walls and the need for an effective lysis protocol compatible with microfluidic platforms. In this work, we present a lysis protocol that can be used to extract genomic DNA from both gram-positive and gram-negative species without interfering with the amplification chemistry. Corynebacterium glutamicum was chosen as a typical gram-positive model and Nostoc sp. as a gram-negative model due to major challenges reported in previous studies. Our protocol is based on thermal and chemical lysis. We consider 80% of single-cell replicates that lead to >5 ng DNA after amplification as successful attempts. The protocol was directly applied to Gloeocapsa sp. and the single cells of the eukaryotic Sphaerocystis sp. and achieved a 100% success rate. View Full-Text
Keywords: bacteria lysis protocol; microalgae lysis; single-cell multiple displacement amplification bacteria lysis protocol; microalgae lysis; single-cell multiple displacement amplification
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Liu, Y.; Schulze-Makuch, D.; De Vera, J.-P.; Cockell, C.; Leya, T.; Baqué, M.; Walther-Antonio, M. The Development of an Effective Bacterial Single-Cell Lysis Method Suitable for Whole Genome Amplification in Microfluidic Platforms. Micromachines 2018, 9, 367.

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