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 endonucleases recognize 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/rest riction.html: Video of restriction enzymes in action! http://www.dnalc.org/resources/animations/rest riction.html

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: Hamemophilus influenzae – 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

8. 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.