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DNA Fingerprinting – Your Bioremediation “Taq”ometer Annemarie L. Douglas1, Michael J. Borda2, and J. Stephen Goudey1 1HydroQual Laboratories Ltd. and 2Golder Associates Inc. Outline ¾ Who I am and who I work for ¾ Explanation of DNA profiling ¾ Examples of how it can be used ¾ Other possible uses ¾ New tests that are being developed HydroQual Laboratories Ltd ¾ HydroQual Laboratories Ltd. (HydroQual) does applied biology for environmental management. ¾ We are a wholly-owned subsidiary of Golder Associates Ltd. ¾ Our talented team of professionals all have degrees and expertise in the natural sciences (microbiology, biochemistry, toxicology, ecology, botany and zoology). ¾ We measure effects for assessing environmental quality. Groups water soil air water quality applied science goldfish DNA Profiling ¾ Sampling ¾ How it is performed ¾ Results All living things have DNA ¾ DNA encodes for all cellular processes ¾ Proteins and enzymes ¾ Cellular machinery ¾ Cellular structure Fingerprinting ¾ Identify individuals within a population ¾ CSI, genetic testing, genealogy ¾ Create a fingerprint of the population ¾ Monitor population changes Conventional Approach Sample Extract bacteria Grow in media Identify Disadvantages ¾ No detection of bacteria that can’t grow ¾ Lots of time - 3 to 21 days or longer Fingerprint Approach Sample Extract DNA Advantages ¾ No culturing = Fast ¾ Sensitive ¾ Small sample size Fingerprint How Do You Fingerprint DNA? Sample Isolate DNA PCR DGGE ¾ Break open the bacteria ¾ Specifically isolate DNA from everything else Polymerase Chain Reaction Sample Isolate DNA PCR DGGE ¾ Need just a small piece of DNA, but lots of it. ¾ What section of the chromosome? ¾ Ribosomal RNA • Involved in protein synthesis • Used for taxonomy conserved ¾ Target population divergent conserved PCR ¾ What’s needed ¾ Thermophilic enzyme = Taq ¾ DNA bases – AGCT ¾ Primers – DNA targets Denaturing Gradient Gel Electrophoresis Sample Isolate DNA PCR DGGE ¾ To separate the PCR DNA ¾ Special gel ¾ Special chemicals to denature DNA ¾ Gradient of chemicals DGGE ¾ A,T – separates at lower conc. ¾ G,C – separates at higher conc. Case Studies ¾ 5 case studies ¾ Range of issues ¾ Demonstrate the capabilities of DNA profiling Study 1 ¾ Effects of nickel on plant growth ¾ 5 types of plants ¾ 11 concentrations of Ni2+ • 0 to 5 g/kg ¾ Soil samples were analyzed by fingerprinting Ni2+ Effects on Plant Growth ¾ Large changes in bacterial population when plants can’t grow ¾ Smaller changes when Ni2+ concentration is lower Study 2 ¾ Biofouling of Bag Filters GW Bag Filter Zero-Valent Iron Wall Air Stripper ¾ Question: Was bacterial growth in the bag filters caused by the remediation system? ¾ Water samples were analyzed by Fingerprinting Biofouling of Bag Filters GW 6 Bag Filter 4, 5 1, 2, 8, 9 Zero-Valent Iron Wall 3 ¾ Bacteria in the bag filters ¾ Don’t appear to be from the incoming GW ¾ Are not leaving the bag filters Air Stripper 7 Study 3 ¾ Contamination with vinyl acetate ¾ Decision: Dig & Dump or Bioremediate ¾ DNA profiling of soil Vinyl Acetate in Soil ¾No bacteria are present in the soil. ¾Bioremediation is not possible. Study 4 ¾ Remediation of chlorinated hydrocarbons with nZVI ¾ 6 wells ¾ 2 treatments with nanoscale zero-valent iron (nZVI) ¾ Fingerprinting of water samples before and after treatment nZVI Remediation ¾ Treatment affected bacterial populations at each well ¾ Bacterial populations got less diverse with treatment 6 months later ¾ Populations are returning to their original state. ¾ Bacterial populations differ by depth. Study 5 ¾ Anaerobic Benzene Bioremediation ¾ Previous treatment with nZVI ¾ Biotreatability study ¾ Addition of N and P ¾ Nitrate – Dechloromonas, etc ¾ Sulfate – Desulfuromonas, etc ¾ Soy protein ¾ Analyze for chemicals and bacteria ¾ DNA profiling for population changes ¾ PCR for detection of specific bacteria Biotreatability Study ¾ N&P alone causes big changes ¾ Soy protein has a short term effect on the bacterial populations ¾ nZVI abrogates some of the nutrient effects Other Uses - DGGE ¾ Bacterial ID – sequencing of fingerprints for definitive ID ¾ Monitoring of microbes, invertebrates, plants, etc. ¾ Baseline studies of soil ¾ Indoor air quality Other Uses - PCR ¾ Microbial ID ¾ Dehalococcoides and other hydrocarbon degraders ¾ Legionella ¾ Cryptosporidium ¾ Biological Activity ID ¾ Culture Maintenance ¾ Plant ID ¾ Source Tracking ¾ Enterococcus ¾ Bacteriodes ¾ Animal Tracking New Products ¾ Quantitative PCR ¾ Species ID and Quantitation ¾ Quantitative RT-PCR ¾ Gene activity measurements ¾ Isolation of RNA TCE Bioremediation RT-PCR cis-Dichloroethene gene 1 TCE Ethene Vinyl Chloride gene 2 ¾ PCR ¾ Detects Dehalococcoides spp. ¾ Detects gene 1 & 2 in the genome ¾ PCR indicates if the bacteria with the correct genes are present but not if the bacteria are alive and degrading TCE ¾ RT-PCR ¾ Detects the expression of gene 1 & 2 ¾ RT-PCR indicates that TCE is already being degraded to ethene Summary ¾ DGGE/PCR ¾ Used for monitoring soil and water ¾ Superior to culturing – speed and sensitivity ¾ More complete picture of in situ populations Questions? ¾ Contact information ¾ Annemarie Douglas ¾ (403) 253-7121 ¾ [email protected]