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      High-Throughput Droplet Digital PCR System for Absolute Quantitation of DNA Copy Number

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      Author(s): † , , † , † , † , † , † , † , † , † , † , † , † , † , † , † , † , † , † , † , † , † , † , † , † , † , † , † , † , † , † , † , † , ‡ , † , † , † , † , †
      Publication date (Electronic):
      Journal: Analytical Chemistry
      Publisher: American Chemical Society

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          Abstract

          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.

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          Rapid planetesimal formation in turbulent circumstellar discs

          Anders Johansen, Jeffrey S. Oishi, Mordecai-Mark Mac Low … (2007)
          The initial stages of planet formation in circumstellar gas discs proceed via dust grains that collide and build up larger and larger bodies (Safronov 1969). How this process continues from metre-sized boulders to kilometre-scale planetesimals is a major unsolved problem (Dominik et al. 2007): boulders stick together poorly (Benz 2000), and spiral into the protostar in a few hundred orbits due to a head wind from the slower rotating gas (Weidenschilling 1977). Gravitational collapse of the solid component has been suggested to overcome this barrier (Safronov 1969, Goldreich & Ward 1973, Youdin & Shu 2002). Even low levels of turbulence, however, inhibit sedimentation of solids to a sufficiently dense midplane layer (Weidenschilling & Cuzzi 1993, Dominik et al. 2007), but turbulence must be present to explain observed gas accretion in protostellar discs (Hartmann 1998). Here we report the discovery of efficient gravitational collapse of boulders in locally overdense regions in the midplane. The boulders concentrate initially in transient high pressures in the turbulent gas (Johansen, Klahr, & Henning 2006), and these concentrations are augmented a further order of magnitude by a streaming instability (Youdin & Goodman 2005, Johansen, Henning, & Klahr 2006, Johansen & Youdin 2007) driven by the relative flow of gas and solids. We find that gravitationally bound clusters form with masses comparable to dwarf planets and containing a distribution of boulder sizes. Gravitational collapse happens much faster than radial drift, offering a possible path to planetesimal formation in accreting circumstellar discs.
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            Long Term Outcomes Following Hospital Admission for Sepsis Using Relative Survival Analysis: A Prospective Cohort Study of 1,092 Patients with 5 Year Follow Up

            Joshua Davis, Vincent He, Nicholas Anstey … (2014)
            Background Sepsis is a leading cause of death in intensive care units and is increasing in incidence. Current trials of novel therapeutic approaches for sepsis focus on 28-day mortality as the primary outcome measure, but excess mortality may extend well beyond this time period. Methods We used relative survival analysis to examine excess mortality in a cohort of 1,028 patients admitted to a tertiary referral hospital with sepsis during 2007–2008, over the first 5 years of follow up. Expected survival was estimated using the Ederer II method, using Australian life tables as the reference population. Cumulative and interval specific relative survival were estimated by age group, sex, sepsis severity and Indigenous status. Results Patients were followed for a median of 4.5 years (range 0–5.2). Of the 1028 patients, the mean age was 46.9 years, 52% were male, 228 (22.2%) had severe sepsis and 218 (21%) died during the follow up period. Mortality based on cumulative relative survival exceeded that of the reference population for the first 2 years post admission in the whole cohort and for the first 3 years in the subgroup with severe sepsis. Independent predictors of mortality over the whole follow up period were male sex, Indigenous Australian ethnicity, older age, higher Charlson Comorbidity Index, and sepsis-related organ dysfunction at presentation. Conclusions The mortality rate of patients hospitalised with sepsis exceeds that of the general population until 2 years post admission. Efforts to improve outcomes from sepsis should examine longer term outcomes than the traditional primary endpoints of 28-day and 90-day mortality.
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              Preparation of Artificial Plasma Membrane Mimicking Vesicles with Lipid Asymmetry

              Qingqing Lin, Erwin London (2014)
              Lipid asymmetry, the difference in lipid distribution across the lipid bilayer, is one of the most important features of eukaryotic cellular membranes. However, commonly used model membrane vesicles cannot provide control of lipid distribution between inner and outer leaflets. We recently developed methods to prepare asymmetric model membrane vesicles, but facile incorporation of a highly controlled level of cholesterol was not possible. In this study, using hydroxypropyl-α-cyclodextrin based lipid exchange, a simple method was devised to prepare large unilamellar model membrane vesicles that closely resemble mammalian plasma membranes in terms of their lipid composition and asymmetry (sphingomyelin (SM) and/or phosphatidylcholine (PC) outside/phosphatidylethanolamine (PE) and phosphatidylserine (PS) inside), and in which cholesterol content can be readily varied between 0 and 50 mol%. We call these model membranes “artificial plasma membrane mimicking” (“PMm”) vesicles. Asymmetry was confirmed by both chemical labeling and measurement of the amount of externally-exposed anionic lipid. These vesicles should be superior and more realistic model membranes for studies of lipid-lipid and lipid-protein interaction in a lipid environment that resembles that of mammalian plasma membranes.
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                Author and article information

                Journal
                Journal ID (nlm-ta): Anal Chem
                Journal ID (publisher-id): ac
                Journal ID (coden): ancham
                Title: Analytical Chemistry
                Publisher: American Chemical Society
                ISSN (Print): 0003-2700
                ISSN (Electronic): 1520-6882
                Publication date (Electronic): 28 October 2011
                Publication date (Print): 15 November 2011
                Volume: 83
                Issue: 22
                Pages: 8604-8610
                Affiliations
                [† ]simpleBio-Rad Laboratories, Inc. , 7068 Koll Center Parkway, Pleasanton, California 94566, United States
                [‡ ]National Genetics Reference Laboratory, Wessex Regional Genetics, simpleSalisbury District Hospital , Odstock, Salisbury, Wiltshire, SP2 8BJ, United Kingdom
                Author notes
                Article
                DOI: 10.1021/ac202028g
                PMC ID: 3216358
                PubMed ID: 22035192
                SO-VID: 2f357534-e6f6-48bd-a51e-5938628e609a
                Copyright © Copyright © 2011 American Chemical Society
                License:

                This is an open-access article distributed under the ACS AuthorChoice Terms & Conditions. Any use of this article, must conform to the terms of that license which are available at http://pubs.acs.org.

                History
                Date received : 03 August 2011
                Date accepted : 05 October 2011
                Date: 28 October 2011
                Date: 15 November 2011
                Funding
                National Institutes of Health, United States
                Categories
                Subject: Article
                Custom metadata
                document-id-old-9 ac202028g
                document-id-new-14 ac-2011-02028g
                ccc-price

                ScienceOpen disciplines: Analytical chemistry
                Data availability:
                ScienceOpen disciplines: Analytical chemistry

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