2 Remember what we know about DNA. What is the monomer of DNA?How do bases pair?What kind of bond is used?
3 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
4 Restriction EnzymesThere are many different restriction enzymes that each cut DNA at different nucleotide sequencesMost will cut the DNA with a staggered cutUsually occurs at a palindrome5'GAATTC3'CTTAAG
5 Sticky endsThe 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
6 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???
8 Restriction Enzymes -Kinds Sticky End- already discussedBlunt EndThese cut the DNA straight across and create blunt ends:CCCGGGGGGCCC
9 Products generated by restriction enzymes COHESIVE END CUTTERS (staggered cuts):Enzyme Recognition Site Ends of DNA After CutEcoRI 5’…GAATTC…3’ 5’…G AATTC…3’3’…CTTAAG…5’ 3’…CTTAA G…5’PstI 5’…CTGCAG…3’ 5’…CTGCA G…3’3’…GACGTC…5’ 3’…G ACGTC…5’BLUNT END CUTTERS (direct cuts):Enzyme Recognition Site Ends of DNA After CutHaeIII 5’…GGCC…3’ 5’…GG CC…3’3’…CCGG…5’ 3’…CC GG…5’
10 Restriction enzymes are named according to the following nomenclature: In case you were curious …Restriction enzymes are named according to the following nomenclature:Ex: EcoRIE = genus Escherichiaco = species coliR = strain RY13I = first enzyme isolated
11 Why would anyone go through the trouble of cutting DNA??? One reason…Recombinant DNABreak down the word…what do you think recombinant means?Other reasons…DNA fingerprinting, gene therapy…
12 DNA that has been cut from one strand of DNA and then inserted into the gap of another piece of DNA that has been broken.The host DNA is often a bacterial cell such as E coli.
13 Bacteria are often used in biotechnology because they have plasmids A plasmid is a circularpiece of DNA that existsapart from thechromosome andreplicates independently of it.
14 The Plasmid is then called a VECTOR What is a vector?Something that is used to transfer genes into a host cellEx’sBacterialplasmidsViruses
15 So how do I isolate a gene of interest? Use a restriction enzyme!!! (duh!)
16 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 other2. Add DNA LIGASE: an enzyme that seals the fragments together
17 What is this organism now called? Transgenic Organism- organisms that contain functional recombinant DNA (rDNA) from a different organism
18 What’s the point?Recombinant DNA has been gaining importance over the last few years, and will become more important as genetic diseases become more prevalent and agricultural area is reduced. Below are some of the areas where Recombinant DNA will have an impact:Better Crops (drought & heat resistance)Recombinant Vaccines (i.e. Hepatitis B)Production of clotting factorsProduction of insulinProduction of recombinant pharmaceuticalsPlants that produce their own insecticidesGerm line and somatic gene therapy
19 RECAP Steps for making a transgenic organism: Locate and isolate the gene of interestCut out the gene and cut the plasmid using the appropriate restriction enzyme
20 3. Insert the desired gene into the plasmid matching up the sticky ends
21 4. Use the enzyme DNA ligase to seal up the sticky ends
22 5. Transfer the vector in the host organism where it will replicate 6. Host organism produces the protein coded for by the recombinant DNA