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WHAT ARE RESTRICTION ENZYMES? Restriction Enzymes – molecular scissors able to cut DNA nuclease Enzyme digests nucleic acids WHAT DNA SEQUENCES DO RESTRICTION ENZYMES CUT? Usually cut DNA at a “palindrome” such as GAATTC. Palindrome – word or phrase when spelled backwords, spells the same word or phrase 5’ 3’ GAATTC Ex. BOB | | | | | | “Restriction site” MADAM I’M ADAM or 3’ CTTAAG 5’ A Toyota! Race fast, safe car. A Toyota “Recognition Sequence” WHAT PARTS OF DNA ARE CUT? RE’s cut DNA’s phosphodiester bonds and hydrogen bonds. WHAT’S LEFT AFTER RESTRICTION ENZYMES CUT DNA? - RE’s generate two different types of “cuts” - Blunt cuts. - Sticky ends WHERE DO RE’S COME FROM? Bacteria “Immune system” to protect against bacteriophages (bacteria-infecting viruses like Lambda). HOW ARE RE’S NAMED? After bacteria which produces them. EcoRI HindIII Genus Escherichia Haemophilus Bacillus Species coli influenzae Strain R d H Order Isolated I III I Recognition Site G^AATTC A^AGCTT BamHI amyloliquefaciens G^GATCC WHAT MUST BE AVAILABLE FOR RESTRICTION ENZYMES TO WORK? All enzymes need the correct: 1. temperature 2. pH “buffer” 3. salt concentration Why are enzymes so needy? Need the same environment as the cell that produced them. WHAT ARE RE’S USED FOR? Genetic engineering – pasting together DNA from two different organisms. Examples: Bacteria with human insulin gene Golden Rice with Vitamin A gene Cows with human milk protein genes Goats with spider silk genes WHAT ARE RE’S USED FOR? Forensics (not always crime scenes) Examples of Transgenic Organisms “Transgenic” – moving genes across species Examples: Bacteria with human insulin gene Golden Rice with Vitamin A gene Cows with human milk protein genes Goats with spider silk genes HOW DO RESTRICTION ENZYMES WORK? Which are more useful in genetic engineering? RE’s that generate sticky ends or ones that produce blunt cuts? STICKY ENDS HOW IS DNA PASTED TOGETHER? Ligase – another enzyme which reconnects phosphodiester bonds.