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Lundin S.,KTH Royal Institute of Technology | Jemt A.,KTH Royal Institute of Technology | Terje-Hegge F.,LingVitae AS | Foam N.,LingVitae AS | And 5 more authors.
PLoS ONE | Year: 2015

Restriction enzymes that recognize specific sequences but cleave unknown sequence outside the recognition site are extensively utilized tools in molecular biology. Despite this, systematic functional categorization of cleavage performance has largely been lacking. We established a simple and automatable model system to assay cleavage distance variation (termed slippage) and the sequence dependence thereof. We coupled this to massively parallel sequencing in order to provide sensitive and accurate measurement. With this system 14 enzymes were assayed (AcuI, BbvI, BpmI, BpuEI, BseRI, BsgI, Eco57I, Eco57MI, EcoP15I, FauI, FokI, GsuI, MmeI and SmuI). We report significant variation of slippage ranging from 1-54%, variations in sequence context dependence, as well as variation between isoschizomers. We believe this largely overlooked property of enzymes with shifted cleavage would benefit from further large scale classification and engineering efforts seeking to improve performance. The gained insights of in-vitro performance may also aid the in-vivo understanding of these enzymes. © 2015 Lundin et al.


Ramachandraiah H.,KTH Royal Institute of Technology | Amasia M.,KTH Royal Institute of Technology | Cole J.,Plarion LTD | Sheard P.,Plarion LTD | And 6 more authors.
Lab on a Chip - Miniaturisation for Chemistry and Biology | Year: 2013

We present a novel "Lab-on-DVD" system and demonstrate its capability for rapid and low-cost HIV diagnostics by counting CD4+ cells isolated from whole blood. We show that a commercial DVD drive can, with certain modifications, be turned into an improved DVD-based laser scanning microscope (DVD-LSM). The system consists of a multi-layered disposable polymer disc and a modified commercial DVD reader with rotational control for sample handling, temperature control for optimized bioassay, a photodiode array for detection, and software for signal processing and user interface-all the necessary components required for a truly integrated lab-on-a-chip system, with the capability to deliver high-resolution images down to 1 μm in size. Using discs modified with antibodies, we specifically captured CD4+ cells from whole blood, demonstrating single cell resolution imaging. The novel integrated DVD platform with sub-micron image resolution brings, for the first time, affordable cellular diagnostic testing to the point-of-care and should be readily applicable at resource-limited settings. © 2013 The Royal Society of Chemistry.

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