04 Recombinant DNA

04 Recombinant DNA - Recombinant DNA technology is ......

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Unformatted text preview: Recombinant DNA technology is ... Recombinant DNA Technology 1. isolating desired DNA fragments 2. joining them in new combinations 3. introducing the newly combined DNA into a living organism. 1. Isolating DNA Fragment Polymerase Chain Reaction Restriction Enzymes Polymerase Chain Reaction Amplify millions of copies of a single piece of DNA in a few hours 1 Cutting DNA with restriction enzymes Restriction endonucleases cleave dsDNA at specific, recognized locations, generally palindromic sequences. The enzymes were discovered in E. coli bacteria in the 1970's. Illustration of endonuclease activity The EcoRI restriction enzyme recognizes the following sequence: -C-A-A-T-T-G-G-T-T-A-A-CThe complementary strand is simply the reverse sequence. A few endonuclease examples 2. Joining DNA in new Combinations Cloning vector: DNA from source organism is cleaved with a restriction endonuclease and inserted into a plasmid DNA construct. Transformation: The resulting DNA construct is introduced into a target host cell. Selection: The cells that carry the construct are identified and grown. http://www.cat.cc.md.us/courses/bio141/lecguide/unit4/genetics/recombination/r ecombinant/enuc.html The protein is produced and harvested. 2 How do you get plasmids? http://www.invitrogen.com figure 17-05.jpg 3. Joining DNA fragments "Sticky" ends formed by restriction enzymes help hold fragments in place temporarily, but the DNA ligase enzyme is necessary to truly "glue" the new dsDNA together. -CTATG AATTCTTGAC-GATACTTAA GAACTGhybridization nick -CTATGAATTCTTGAC-GATACTTAAGAACTGnick DNA ligase (ligation) -CTATGAATTCTTGAC-GATACTTAAGAACTG- How do you know if it worked? Agarose Gel Electrophoresis DNA Sizing Tutorial 16.1 http://www.icampus.ucl.ac.be/SBIM2520/document/genemol/electrotext.html 3 ...
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