Development of an automated DNA purification module using a micro-fabricated pillar chip

We present a fully automated DNA purification module comprised of a micro-fabricated chip and sequential injection analysis system that is designed for use within autonomous instruments that continuously monitor the environment for the presence of biological threat agents. The chip has an elliptical flow channel containing a bed (3.5 &times; 3.5 mm) of silica-coated pillars with height, width and center-to-center spacing of 200, 15, and 30 &micro;m, respectively, which provides a relatively large surface area (ca. 3 cm2) for DNA capture in the presence of chaotropic agents. We have characterized the effect of various fluidic parameters on extraction performance, including sample input volume, capture flow rate, and elution volume. The flow-through design made the pillar chip completely reusable; carryover was eliminated by flushing lines with sodium hypochlorite and deionized water between assays. A mass balance was conducted to determine the fate of input DNA not recovered in the eluent. The device was capable of purifying and recovering Bacillus anthracis genomic DNA (input masses from 0.32 to 320 pg) from spiked environmental aerosol samples, for subsequent analysis using polymerase chain reaction-based assays.<br /

Underwater Application of Quantitative PCR on an Ocean Mooring

The Environmental Sample Processor (ESP) is a device that allows for the underwater, autonomous application of DNA and protein probe array technologies as a means to remotely identify and quantify, in situ, marine microorganisms and substances they produce. Here, we added functionality to the ESP through the development and incorporation of a module capable of solid-phase nucleic acid extraction and quantitative PCR (qPCR). Samples collected by the instrument were homogenized in a chaotropic buffer compatible with direct detection of ribosomal RNA (rRNA) and nucleic acid purification. From a single sample, both an rRNA community profile and select gene abundances were ascertained. To illustrate this functionality, we focused on bacterioplankton commonly found along the central coast of California and that are known to vary in accordance with different oceanic conditions. DNA probe arrays targeting rRNA revealed the presence of 16S rRNA indicative of marine crenarchaea, SAR11 and marine cyanobacteria; in parallel, qPCR was used to detect 16S rRNA genes from the former two groups and the large subunit RuBisCo gene (rbcL) from Synecchococcus. The PCR-enabled ESP was deployed on a coastal mooring in Monterey Bay for 28 days during the spring-summer upwelling season. The distributions of the targeted bacterioplankon groups were as expected, with the exception of an increase in abundance of marine crenarchaea in anomalous nitrate-rich, low-salinity waters. The unexpected co-occurrence demonstrated the utility of the ESP in detecting novel events relative to previously described distributions of particular bacterioplankton groups. The ESP can easily be configured to detect and enumerate genes and gene products from a wide range of organisms. This study demonstrated for the first time that gene abundances could be assessed autonomously, underwater in near real-time and referenced against prevailing chemical, physical and bulk biological conditions

Soil chemistry effects and flow prediction in remediation of soils by electric fields

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 1996.Includes bibliographical references (p. 121-123).by John Mason Dzenitis.Ph.D

Stability of qPCR assays held at ambient temperature on the MFB.

<p>Stability of qPCR assays held at ambient temperature on the MFB.</p

Environmental conditions and real time results from the ESP deployed at station M0, Monterey Bay from May 14–June 11, 2009.

<p>Hourly winds measured at station M2 that indicate regional forcing (A). Colored bar indicates strong upwelling conditions (blue), relaxation-reversal of upwelling favorable winds (white), and conditions dominated by local physical processes (black). The dashed lines show the lag response between the atmospheric and oceanographic data. CTD data (binned to 3 hours) from the moored ESP included chlorophyll (B), salinity, nitrate and temperature (C). Only a subset of the bacterioplankton groups detected on the ribotype arrays are shown (D). Array signals for SAR11, marine crenarchaea, and marine <i>Roseobacter</i> were converted to ng target rRNA per mL lysate using standard curves <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022522#pone.0022522-Preston1" target="_blank">[35]</a>. The average raw pixel intensity from array probe spots is presented for marine cyanobacteria. Marine crenarchaeal 16S rRNA, SAR11 16S rRNA and <i>Synechococcus rbcL</i> genes, expressed as copies per L seawater (E). Starred data points in panel E represent genes detected but unquantifiable (Ct<10² standard) and Xs mark dates when the negative controls were run.</p