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Micromachines 2017, 8(2), 62; doi:10.3390/mi8020062

Droplet Microfluidics Approach for Single-DNA Molecule Amplification and Condensation into DNA-Magnesium-Pyrophosphate Particles

Institute of Biotechnology, Vilnius University, Saulėtekio Avenue 7, LT-02571 Vilnius, Lithuania
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Academic Editor: Andrew J. deMello
Received: 7 December 2016 / Revised: 3 February 2017 / Accepted: 14 February 2017 / Published: 20 February 2017
(This article belongs to the Special Issue Droplet Microfluidics: Techniques and Technologies, Volume II)
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Abstract

Protein expression in vitro has broad applications in directed evolution, synthetic biology, proteomics and drug screening. However, most of the in vitro expression systems rely on relatively high DNA template concentrations to obtain sufficient amounts of proteins, making it harder to perform in vitro screens on gene libraries. Here, we report a technique for the generation of condensed DNA particles that can serve as efficient templates for in vitro gene expression. We apply droplet microfluidics to encapsulate single-DNA molecules in 3-picoliter (pL) volume droplets and convert them into 1 μm-sized DNA particles by the multiple displacement amplification reaction driven by phi29 DNA polymerase. In the presence of magnesium ions and inorganic pyrophosphate, the amplified DNA condensed into the crystalline-like particles, making it possible to purify them from the reaction mix by simple centrifugation. Using purified DNA particles, we performed an in vitro transcription-translation reaction and successfully expressed complex enzyme β-galactosidase in droplets and in the 384-well format. The yield of protein obtained from DNA particles was significantly higher than from the corresponding amount of free DNA templates, thus opening new possibilities for high throughput screening applications. View Full-Text
Keywords: in vitro expression; DNA condensation; droplet microfluidics; phi29 DNA polymerase in vitro expression; DNA condensation; droplet microfluidics; phi29 DNA polymerase
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Zubaite, G.; Simutis, K.; Galinis, R.; Milkus, V.; Kiseliovas, V.; Mazutis, L. Droplet Microfluidics Approach for Single-DNA Molecule Amplification and Condensation into DNA-Magnesium-Pyrophosphate Particles. Micromachines 2017, 8, 62.

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