High-throughput droplet digital PCR system for absolute quantitation of DNA copy number.

Digital PCR enables the absolute quantitation of nucleic acids in a sample. The lack of scalable and practical technologies for digital PCR implementation has hampered the widespread adoption of this inherently powerful technique. Here we describe a high-throughput droplet digital PCR (ddPCR) system that enables processing of ~2 million PCR reactions using conventional TaqMan assays with a 96-well plate workflow. Three applications demonstrate that the massive partitioning afforded by our ddPCR system provides orders of magnitude more precision and sensitivity than real-time PCR. First, we show the accurate measurement of germline copy number variation. Second, for rare alleles, we show sensitive detection of mutant DNA in a 100,000-fold excess of wildtype background. Third, we demonstrate absolute quantitation of circulating fetal and maternal DNA from cell-free plasma. We anticipate this ddPCR system will allow researchers to explore complex genetic landscapes, discover and validate new disease associations, and define a new era of molecular diagnostics.

Lignolytic Enzymes of a Mushroom Stereum ostrea Isolated from Wood Logs.

Production of lignolytic enzymes by the mushroom fungus Stereum ostrea in liquid medium under conditions of vegetative growth was examined for 10 days in comparison to the reference culture Phanerochaete chrysosporium. Though growth and secretion of extracellular protein by S. ostrea were comparable to those of P. chrysosporium, yields of laccase enzyme by S. ostrea were higher than laccase titres of P. chrysosporium by more than 2 folds on the peak production time interval (IVth day of incubation). S. ostrea yielded titres of 25 units of laccase/ml as against 8.9 units of laccase/ml on the IVth day of incubation. Stereum ostrea also exhibited activities of other lignolytic enzymes, lignin peroxidase (LiP) and manganese peroxidase (MnP), higher than the reference culture. Growth of S. ostrea on the medium in the presence of Remazol orange 16 resulted in the decolourisation of dye, confirming the presence of lignolytic enzymes. S. ostrea appears to be a promising culture with complete lignolytic system.