BIO172W08Lec23CT - Biology 172 Lecture 23: DNA Technology I...

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Lecture 23: DNA Technology I March 10, 2008 Today’s Outline 1. Recombinant DNA Technology 2. Plasmids Restriction enzymes Cloning DNA cDNA libraries Nucleic acid hybridization 3. Analyzing DNA PCR DNA sequencing Genetic markers Genetic testing Announcements This week in Discussion: DNA Cloning Biology 172
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DNA Technology is Genetic Engineering Ability to manipulate genes within and across organisms = genetic recombination (recombinant DNA technology) Cloning DNA in bacterial plasmids (use E. coli to make many copies of foreign DNA) Relies on bacterial enzymes that cut DNA at specific places ( restriction enzymes ) and other enzymes that paste DNA sequences together ( ligases ) Another technique to amplify DNA is the polymerase chain reaction (PCR) Countless practical uses for biotechnology (medicine, industry, agriculture, forensics)
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Plasmids occur naturally in bacteria Small, circular molecules of DNA distinct from the chromosome Often contain genes for antibiotic resistance or responses to environment Transferred from one bacterium to another by conjugation (a type of horizontal gene transfer from one organism to another)
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Cloning DNA = use host cell ( E. coli ) to make many copies of a fragment of recombinant DNA
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Restriction endonucleases Bacterial enzymes that cut double stranded DNA at specific base sequence (recognition site ) Used by bacteria to destroy infecting viral DNA
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Sticky Ends and Blunt Ends Restriction site is often a palindrome = reads the same in either direction “Was it a cat I saw?” Sticky ends EcoRI 5’ GAATTC 3’ CTTAAG G AATTC CTTAA G Blunt ends EcoRV 5’ GGATCC GGA TTC CCT AAG 3’ CCTAGG
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Use same RE ( e.g., EcoRI) to cut both plasmid vector and foreign DNA 2. Sticky ends base pair (directional cloning ) 3. Use DNA ligase to join
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BIO172W08Lec23CT - Biology 172 Lecture 23: DNA Technology I...

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