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Recombinant DNA Techonology

Recombinant DNA Techonology. 4.3. Introduction.

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Recombinant DNA Techonology

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  1. Recombinant DNA Techonology 4.3

  2. Introduction • If you pay any attention at all to the news, you cannot avoid stories about biotechnology: sequencing a genome, identifying a gene, analyzing ancient DNA, fingerprinting DNA, cloning something, genetic engineering, etc. Have you ever wondered, “How do they do that?”

  3. Making Recombinant DNA (rDNA) • Enzymes called restriction endonucleasesrecognize specific DNA base sequences and cut the DNA at or near the recognition sequence. • Produced by bacteria. • When added to a t.t. containing DNA, they enzyme will cut the DNA molecules at every occurrence of its recognition sequence. • http://www.dnalc.org/resources/animations/restriction.html: Video of restriction enzymes in action!

  4. Palindromes • Most commonly used restriction enzymes recognize palindromes. • Base sequences in which both strands read the same in the 5’ to 3’ direction. • Ex. 5’ GAATTC 3’ and the complimentary strand would read from 3’ to 5’ (left to right) as follows: 3’ CTTAAG 5’.

  5. How many restriction endonucleases are there? • Little over 100. • Names indicate the organism from which they were purified. • EcoRI: Escherichia coli • HindIII: Hamemophilusinfluenzae • BamHI: Bacillus amyloliquefaciens • Each enzyme bind to & cut at specific DNA base sequences every time. • Cut pieces are called restriction fragments.

  6. Stick & Blunt Ends

  7. Now what? • Now we have small pieces of (restriction fragments) DNA in which we could: • Electrophorese the fragments. • Glue 2 or more fragments from different organisms back together. Glue = DNA ligase. Now the DNA is called recombinant DNA! • Ex. If your goal is to insert a new gene into a microorganism, plant, or animal to clone a gene.

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