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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| Veröffentlicht in: | ACS synthetic biology 2021-09, Vol.10 (9), p.2179-2186 |
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| container_title | ACS synthetic biology |
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| creator | Ueno, Hiroshi Sawada, Hiroki Soga, Naoki Sano, Mio Nara, Seia Tabata, Kazuhito V Su’etsugu, Masayuki Noji, Hiroyuki |
| description | 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. |
| doi_str_mv | 10.1021/acssynbio.0c00584 |
| format | Article |
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| language | eng |
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| source | MEDLINE; ACS Publications |
| subjects | Cell-Free System DNA - genetics DNA - metabolism DNA Primers - metabolism Lipid Droplets - chemistry Nucleic Acid Amplification Techniques - methods |
| title | Amplification of over 100 kbp DNA from Single Template Molecules in Femtoliter Droplets |
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