Amplification of over 100 kbp DNA from Single Template Molecules in Femtoliter Droplets

Reconstitution of the DNA amplification system in microcompartments is the primary step toward artificial cell construction through a bottom-up approach. However, amplification of >100 kbp DNA in micrometer-sized reactors has not yet been achieved. Here, implementing a fully reconstituted repliso...

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Bibliographic Details
Published in:ACS synthetic biology 2021-09, Vol.10 (9), p.2179-2186
Main Authors: Ueno, Hiroshi, Sawada, Hiroki, Soga, Naoki, Sano, Mio, Nara, Seia, Tabata, Kazuhito V, Su’etsugu, Masayuki, Noji, Hiroyuki
Format: Article
Language:English
Subjects:
Cell-Free System
DNA - genetics
DNA - metabolism
DNA Primers - metabolism
Lipid Droplets - chemistry
Nucleic Acid Amplification Techniques - methods
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Summary:Reconstitution of the DNA amplification system in microcompartments is the primary step toward artificial cell construction through a bottom-up approach. However, amplification of >100 kbp DNA in micrometer-sized reactors has not yet been achieved. Here, implementing a fully reconstituted replisome of Escherichia coli in micrometer-sized water-in-oil droplets, we developed the in-droplet replication cycle reaction (RCR) system. For a 16 kbp template DNA, the in-droplet RCR system yielded positive RCR signals with a high success rate (82%) for the amplification from single molecule template DNA. The success rate for a 208 kbp template DNA was evidently lower (23%). This study establishes a platform for genome-sized DNA amplification from a single copy of template DNA with the potential to build more complex artificial cell systems comprising a large number of genes.
ISSN:2161-5063
2161-5063
DOI:10.1021/acssynbio.0c00584