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Lecture 16 Bio325 Fall 07

Lecture 16 Bio325 Fall 07 - The tools now used in molecular...

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1 The tools now used in molecular biology arose from bacterial genetics: restriction enzymes: some strains of E. coli were found to be resistant to bacteriophage infection in these strains, the bacteriophage DNA inside the E. coli cells was found to be cut into many pieces the enzyme that degrades the phage DNA is a restriction enzyme and acts to protect the cell from foreign DNA Fig. A, p. 306 E. coli strains with restriction enzymes also have modification enzymes which methylate the chromosomal DNA in order to protect it from digestion by the restriction enzyme: every restriction enzyme is specific to a particular DNA sequence bricker.tcnj.edu/ tech/le3/ResMod.jpg
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2 Every restriction enzyme recognizes and cuts a specific DNA sequence The sequence can be between 4 and 16 base-pairs long (majority of recognition sequences are 4 to 8 bp) Most recognition sites are palindromic Fig. 9.2 Recombinant DNA technology: “Cloning” ~ genetic engineering 1973; Boyer and Cohen realized that bacterial plasmids could be manipulated to include DNA from other plasmids or even other organisms. Once in a plasmid, the DNA would be replicated along with the bacterial chromosome. Therefore, an unlimited supply of the DNA could be made just be growing more and more of the bacterial strain containing the plasmid. Why do we use plasmids to do genetic engineering? • plasmids are small (~1,500 to 20,000 bp) • they are nonessential to the bacterial host
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3 Steps in cloning of DNA fragments: 1. cut vector with restriction enzyme(s) and cut insert DNA with restriction enzyme(s) 2. Incubate with ligase to join the ends. 3. Transform E. coli and select for antibiotic resistance gene (or other selectable marker) present on the vector 4. Grow colonies in liquid cultures, isolate plasmid, test for presence of insert Fig. 9.7 Natural sources of DNA generally do not provide enough material for cloning. If some sequence information is known, then a DNA fragment can be amplified from small amounts of a native source by PCR (Polymerase Chain Reaction) Where does the insert DNA come from? • cut out of another plasmid or cloned DNA source • from a native source (i.e. tissue, blood, any source of cellular DNA) components= DNA substrate DNA primers (short single-stranded DNA’s homologous to the DNA substrate) to initiate polymerization deoxynucleotides Heat-stable DNA polymerase 1. denaturation 2. annealing 3. extension 20 - 40 of these cycles: Fig. 9.16
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