A completely automated sample preparation instrument and consumable device for isolation and purification of nucleic acids.

Molecular diagnostic analysis and life science studies are dependent on the ability to effectively prepare samples for analysis. We report the development of a system that enables robust sample preparation of nucleic acids. To enable completely automated sample preparation, a consumable cartridge and consumable module system were developed to emulate every step of the sample preparation process. This included enzyme and reagent addition, temperature-controlled incubations, noncontact mixing of enzymes and reagents, buffer exchanges, and sample elution. Using this system, completely automated methods were developed for the purification of viral RNA and DNA from plasma and whole blood and of bacterial genomic DNA from water and whole blood. Extracted nucleic acids were detected and quantified using real-time PCR. The data indicate that automated viral DNA extraction was more efficient than sample extractions performed using a manual process, whereas automated total RNA extraction from the same samples was equivalent to controls. Additionally, we found that the process for bacterial genomic DNA extraction from either water or whole blood was equivalent to the manual extraction processes. We conclude the instrument, consumable cartridge, and reagent system enables easy, cost-effective, and robust sample preparation regardless of the experience of the operator.

Expression of the DNA repair enzyme, photolyase, in developmental tissues and larvae, and in response to ambient UV-R in the Antarctic sea urchin Sterechinus neumayeri.

Gene expression of the DNA repair enzyme, photolyase (E.C. 4.1.99.3) was examined in the gonads, eggs, embryos and larval stages of the Antarctic sea urchin, Sterechinus neumayeri. Partial sequencing of the gene revealed two highly conserved regions, including a 300 bp region representing the binding site for the cofactor flavin adenine dinucleotide. The second 1200 bp region, likely representing a second light-harvesting cofactor binding site, was identified in a second sea urchin species, Strongylocentrotus frascicanus. Probes for photolyase were developed from the shorter sequence, and expression in sea urchin developmental tissue and stages, and in response to in situ exposure to ultraviolet radiation was quantified using PCR and RT-qPCR, with concentrations of photolyase normalized to actin concentrations. Photolyase was expressed in all tissues and developmental stages examined. In controlled field-based experiments in McMurdo Sound, Antarctica, we found evidence of both constitutive expression of photolyase and induction in response to in situ exposure of embryos to UV-R. Induction of photolyase was observed in response to greater ambient UV-R (such as shallower water depths or sea ice-free regions).