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Chapter 16

Chapter 16 - DNA Technologies and Genomics.docx

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BIOL 204
Dr.Roland Treu

CHAPTER 16 DNA TECHNOLOGIES AND GENOMICSVenters survey of genetic diversity in the ocean is an example of the emerging field of metagenomics in which DNA from an entire community of organisms in a particular niche is harvested collectively sequenced and analyzed using DNA technologiesThis approach is significant bc until very recently our understanding of the genetics of the microbial world was based almost exclusively on the very small proportion of species that can be cultivated in the laboratoryow tools of modern metagenomics we gain access to genomes of a whole new world of previously inaccessible organismsoUse of such genes for practical purposesgenetic engineeringGenetic engineering is latest addition to broad area of biotechnology which is any technique applied to biological systems or living organisms to makemodify productsprocesses for specific purposeoIncludes manipulations that dont involve DNA technologies eg use of yeast to brew beer and bake bread and use of bacteria to make yogurt and cheese161 DNA CloningMethod for producing many copies of piece of DNA piece referred to as gene of interestCloned genes are used to basic research to find out about their biological functionsoAlso in applied research eg in case of genes that encode proteins of pharmaceuticalclinical importanceCommon method for cloning gene of interest cloning DNA fragments in bacterial plasmid161a Bacterial Enzymes Restriction EndonucleasesKey to DNA cloning is specific joining of 2 DNA molecules from diff sources eg genomic DNA fragmentbacterial plasmidoMade possible in part by bacterial enzymes restriction endonucleases aka restriction enzymesoThey recognize short specific DNA sequences restriction sites typically 48 bases long and cut DNA at specific locations win those sequences restriction fragmentsorestriction refers to normal role inside bacteria in which enzymes defined against viral attack by breaking down restricting DNA molecules of infecting viruses100s of restriction enzymes have been IDd each one cutting DNA at specific restriction siteEx restriction site for restriction enzyme EcoRImost used in cloningoMost restriction sites are symmetrical in that sequence of nucleotides read in 5 to 3 direction on one strand is same as sequence read in 5 to 3 direction on complementary strandoRestriction enzymes cleave the sugarphosphate backbones of DNA to produce DNA fragments with singlestranded ends Ends are called sticky ends because short singlestranded regions can form hydrogen bonds w complementary sticky ends on any other DNA molecules cut with same enzyme161b Bacterial Plasmids Illustrate the Use of Restriction Enzymes in CloningBacterial plasmids used for cloning are examples of cloning vectorsDNA molecules into which DNA fragment can be inserted to form recombinant DNA molecule for cloningCommonly plasmid cloning vectors engineered to contain 2 genes useful in final steps of cloning experiment for IDg bacteria that have recombinant plasmids from those that do notRamp gene encodes enzyme that breaks down antibiotic ampicillinRoWhen plasmid introd into E Coli and amp gene is expressed bacteria become resistant to ampicillinLacZ gene encodes galactosidase whic hydrolyzes sugar lactoseoRestriction sites located win lacZ gene but dont alter genes functionFor a given cloning experiment one of these restriction sites is chosenCloning a Gene of Interest
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