Published on October 15, 2007
Discover the Microbes Within:Impacts of DNA-based technologies and PCR basics: Discover the Microbes Within: Impacts of DNA-based technologies and PCR basics Seth Bordenstein Marine Biological Laboratory May 5, 2007 Slide2: 0.1 changes per nt EUBACTERIA ARCHAEBACTERIA EUCARYA Homo Coprinus Paramecium Naegleria pSL 22 pSL 50 Thermofilum Methanospirillum Methanobacterium Thermococcus Thermotoga Thermus Synechococcus Bacilllus Cytophaga Chlorobium Wolbachia E. coli Riftia macroscopic organisms Zea Porphyra Dictyostelium Entamoeba Euglena Trypanosoma Physarum Encephalitozoon Vairimorpha Giardia Hexamita Tritrichomonas pJP 78 pJP 27 marine group1 pSL 12 pSL 4 Thermoproteus Sulfolobus Haloferax Methanosarcina Methanococcus Methanopyrus EM 17 Aquifex Thermomicrobium chloroplast Epulopiscium mitochondria Chromatium origin C. Woese Bacteria Fun Facts: Bacteria Fun Facts Most microbes do not cause disease! 1 gram of soil = 10 million bacteria More bacteria in your mouth than there are people in the world # beneficial bacteria in human intestine = # of cells in human body Oldest forms of life on earth are bacteria (3.8 billion years ago) Bacteria make up most of the biomass on earth, but only 1% have been cultured Classical microbiology – phenotypic approach: Classical microbiology – phenotypic approach Culture Gram stain Shapes Molecular microbiology – genotypic approach: Molecular microbiology – genotypic approach Identifies genes More accurate, objective, and reproducible results Identifies unculturable bacteria Eliminates special growth requirements DNA sequence data are more easily shared, transported, and databased Rapid diagnosis Genes and Genomes: fun facts: Genes and Genomes: fun facts Bacteria have small genomes (1-10 million base pairs vs. 3 billion base pairs in human) 1995: Entire genome of Haemophilus influenzae was sequenced Two years later: 12 genomes sequenced April 5, 2007: 620 genomes sequenced (510 are from bacteria) and 2135 ongoing (1305 from bacteria) Slide7: Buchnera (0.64 Mb) Blochmannia (0.75 Mb) Wolbachia (1.27 Mb) Wigglesworthia (0.70 Mb) Slide8: Endosymbionts are the smallest, most AT-rich bacterial genomes known Wernegreen 2002 Insect endosymbionts How do we go from here…..: How do we go from here….. …to studying the microbes within?: Insect nuclei Wolbachia Insect Egg …to studying the microbes within? Number of papers with Wolbachia in title: Number of papers with Wolbachia in title Nobel prize awarded for PCR PCR detection of Wolbachia in ~20% of all insects PCR Introduction: PCR Introduction Polymerase Chain Reaction (PCR) allows scientists to amplify minutes amount of a specific DNA sequence The DNA is copied millions of times in just a few hours, so that the DNA becomes easy to detect, study, and use in any fashion. PCR is revolutionizing research in bacterial diversity, genetic disease diagnosis, forensic medicine, and molecular evolution Invented by Dr. Kary Millis in 1983 (Nobel Prize in Chemistry in 1993) One of the most widely used biotechnology techniques in biological research. PCR is the method of choice for symbiont detection. Basics of PCR: Basics of PCR Template DNA - the starting DNA of interest. High temperature denatures template DNA into single strands and synthetic sequences of ssDNA (20-30 nucleotides) serve as primers Two different primers are used to bracket the target gene to be amplified DNA polymerase copies the complimentary strand starting at the primer. In one cycle, two identical strands are made. To perform your PCR today: To perform your PCR today Small quantity of DNA Primers Buffered solution containing DNA polymerase Four base pairs of DNA Cofactor MgCl2 All in test tube Buffered solution containing DNA polymerase Four base pairs of DNA Cofactor MgCl2 Small quantity of DNA Primers Target molecule: 16S rRNA: Target molecule: 16S rRNA Small ribosomal subunit involved in mRNA translation process Ancient molecule, functionally constant, universally distributed Microbial diversity - identify unknown bacterium to genus or species levels Present in bacterial symbionts; eukaryote has very divergent copy that is named 18S rRNA PCR Animation: PCR Animation Two Key Innovations for Success of PCR: Two Key Innovations for Success of PCR Heat-stable DNA polymerase isolated from bacterium Thermus aquaticus which inhabits hot springs Polymerase remains active despite being heated many times DNA thermal cyclers – a computer that controls repetitive temperature changes required for PCR 70C hot springs in Yellowstone National Park Example of a thermal cycler from MJ Research PCR and Pop Culture: PCR and Pop Culture “Jurassic Park” and “CSI” Some fun PCR facts to share with your students: …PCR has been used to amplify DNA from… a preserved quagga (a zebra relative that became extinct 100 years ago) crime scenes (e.g., O.J.) eight-celled human preembryos, to detect cystic fibrosis the brain of a 7000 year old American mummy patients for disease diagnosis Our goal: Determine which of your insects harbor the Wolbachia parasite?: Our goal: Determine which of your insects harbor the Wolbachia parasite? DNA extraction: PCR: Gel electrophoresis: One day till we discover the microbes within!: One day till we discover the microbes within!