Wednesday, February 29, 2012

Chromium DNA, and Soil Microbial Communities. DNA Working and RNA

Chromium DNA, and Soil Microbial Communities. DNA Working and RNA

The experiments were conducted on a previously uncontaminated soil amended with various levels of hexavalent Cr. Only 2-3% of the Cr was bio available 10 days after addition of the metal. Even though Cr bioavailability decreased rapidly, the bacterial community richness, as measured using denaturing gradient gel electrophoresis (DGGE), was reduced for both the 200 and 1000 mg kg-1 Cr treatments. One year after contamination, bacterial community richness had not returned to pre-disturbance levels. However, the fungal community had increased richness for the Cr-contaminated soils that was maintained for the duration of experiment. Changes in bacterial community richness were significantly related to both the total amount of Cr added and Cr bioavailability. From this research, there appears to be an antagonistic relationship between the fungal and bacterial communities stressed by Cr, which is significantly related to the total metal in the system. Due to technical problems with the amplification of DNA isolated from these chromium containing soils, I hypothesized that Cr was extracted along with the DNA isolated directly from soil. Using size exclusion chromatography with inductively coupled plasma mass spectrometry (SEC-ICP-MS), I determined the DNA extracts contained Cr. However, Cr was not free in solution indicating that inhibition of PCR was related to the environmental DNA samples containing low levels of Cr (0.5-0.7 ng g-1). To further understand the impact of Cr bound to DNA, DNA from an eight member model bacterial community was extracted in the presence of varying concentrations of Cr and community structure was measured using DGGE. The DGGE profiles of the DNA extracted in the presence of Cr had decreases in the number of bands corresponding to the model community, as well as decreases in the intensity of individual bands, as Cr concentrations increased. These results indicate that PCR-based molecular analyses of metal containing DNA may indicate altered community profiles that do not reflect actual changes in the community.

Bioelectronic DNA detection involves forming an electronic circuit mediated by nucleic acid hybridization and it serves as the basis for a DNA detection system called eSensor™ [1-4]. This system uses low-density DNA chips containing electrodes coated with DNA capture probes. Target DNA present in the sample hybridizes specifically both to capture probes and ferrocene labeled signal probes in solution thereby generating an electric current. Currente Sensor DNA chips contain as many as 36 electrodes for simultaneous detection of multiple pathogens from a single sample.

Many pathogens cause both acute and chronic disease at relatively low copy number and may be difficult or impossible to propagate in culture. Thus, most pathogen detection systems rely on nucleic acid amplification by using polymerase chain reaction (PCR). One highly effective amplification strategy targets conserved sequences among the family of organisms of interest. Such broad-range PCR strategies have been used to identify and characterize several known and previously uncharacterized bacteria [5,6] and viruses [7,8]. In order to maximize the utility of these effective pathogen nucleic acid amplification systems, amplification needs to be coupled with rapid, sensitive, and specific detection. Bioelectronic DNA detection by use of the eSensor chip might fulfill this need.

Human papillomaviruses (HPV) serve as an ideal model system for determining the efficiency and feasibility of eSensor DNA detection technology since there are at least 30 distinct genital HPV types that can be effectively amplified with broad-range consensus PCR primers. We designed two eSensor chips, each containing 14 probes specific for the conserved L1 region of the HPV genome. We evaluated clinical cervical cytology samples known to contain one or more HPV types. The eSensor DNA detection platform successfully detected the correct HPV type in most of these clinical samples, demonstrating that the system provides a rapid, sensitive, specific, and economical approach for multiple-pathogen detection and identification from a single sample.Background We used human papillomaviruses (HPV) as a model system to evaluate the utility of a nucleic acid, hybridization-based bioelectronic DNA detection platform (eSensor) in identifying multiple pathogens.