Restriction Enzymes Solon Schools限制性酶索伦学校.pptVIP

Restriction Enzymes Remember what we know about DNA. What is the monomer of DNA? How do bases pair? What kind of bond is used? Restriction Enzymes Aka Restriction Endonucleases What macromolecule do you think they are made of? Right, they are PROTEINS that cut strands of DNA at specific nucleotide sequences Restriction Enzymes There are many different restriction enzymes that each cut DNA at different nucleotide sequences Most will cut the DNA with a staggered cut Usually occurs at a palindrome Sticky ends The staggered cuts leave the DNA with end pieces “sticking off” We call these “sticky ends” These exposed N-bases will want to join with other complimentary exposed bases What if??? What do you predict could happen if two pieces of DNA are cut with the same restriction enzyme??? YES! They will have the same “sticky ends” How could we use this??? Restriction Enzymes -Kinds Sticky End- already discussed Blunt End These cut the DNA straight across and create blunt ends: CCCGGG GGGCCC Restriction enzymes are named according to the following nomenclature: Ex: EcoRI E = genus Escherichia co = species coli R = strain RY13 I = first enzyme isolated Why would anyone go through the trouble of cutting DNA??? One reason… Recombinant DNA Break down the word…what do you think recombinant means? Other reasons…DNA fingerprinting, gene therapy… The Plasmid is then called a VECTOR What is a vector? Something that is used to transfer genes into a host cell Ex’s Bacterial plasmids Viruses So how do I isolate a gene of interest? Use a restriction enzyme!!! (duh!) What next??? Once the gene is isolated, how do we join it with the organism’s DNA? 1. Cut the organism’s DNA with the same restriction enzyme…why? The sticky ends will naturally be attracted to each other 2. Add DNA LIGASE: an enzyme that seals the fragments together What is this organism now called? Transgenic Organism- organisms that contain functional recombinant DNA (rDNA) from a different