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Recombinant DNA and Plasmid Notes

Recombinant DNA and Plasmid Notes. February 9, 2011. Remember DNA?. What is the monomer of DNA? Nucleotides How do bases pair? A – T C – G What kind of bond is used? Hydrogen bonds between nitrogen bases. I. Restriction Enzymes. AKA Restriction Endonucleases

deborah-mosley
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Recombinant DNA and Plasmid Notes

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  1. Recombinant DNA and Plasmid Notes February 9, 2011

  2. Remember DNA? • What is the monomer of DNA? • Nucleotides • How do bases pair? • A – T • C – G • What kind of bond is used? • Hydrogen bonds between nitrogen bases

  3. I. Restriction Enzymes • AKA Restriction Endonucleases • What macromolecule do you think they are made of? • They are PROTEINS that cut strands of DNA at specific nucleotide sequences

  4. Restriction Enzymes (cont.) • 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: a sequence of units that can be read the same way in either direction 5‘…GAATTC…3’ 3‘…CTTAAG…5’

  5. D. 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

  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???

  7. E. Types of Restriction Enzymes • Sticky End- already discussed • Blunt End • These cut the DNA straight across and create blunt ends: CCC GGG GGG CCC

  8. F. Products generated by restriction enzymes 1. COHESIVE END CUTTERS (staggered cuts): EnzymeRecognition SiteEnds of DNA After Cut 5’…G AATTC…3’ 3’…CTTAA G…5’ 5’…GAATTC…3’ 3’…CTTAAG…5’ EcoRI 5’…CTGCA G…3’ 3’…G ACGTC…5’ PstI 5’…CTGCAG…3’ 3’…GACGTC…5’ 2. BLUNT END CUTTERS (direct cuts): EnzymeRecognition SiteEnds of DNA After Cut 5’…GG CC…3’ 3’…CC GG…5’ 5’…GGCC…3’ 3’…CCGG…5’ HaeIII

  9. 1. Restriction enzymes are named according to the following nomenclature: G. Restriction Enzyme Naming Ex: EcoRI • E = genus Escherichia • co = species coli • R = strain RY13 • I = first enzyme isolated

  10. 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…

  11. II. Recombinant DNA • Recombinant DNA: 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.

  12. B. Bacterial Structure • Bacteria are often used in biotechnology because they have plasmids • A plasmid is a circular piece of DNA that exists apart from the chromosome and replicates independently of it. • A plasmid is therefore called a VECTOR.

  13. C. Vectors • What is a vector? • Something that is used to transfer genes into a host cell • Examples • Bacterial plasmids • Viruses

  14. D. Isolating Genes • Must isolate the gene of interest first before you insert it into the plasmid • How do you do this? • Use a restriction enzyme!!!

  15. E. Final Steps of Making Recombinant DNA • Once the gene is isolated, have to cut the organism’s DNA with the same restriction enzyme…why? • The sticky ends will naturally be attracted to each other • Add DNA LIGASE: enzyme that seals the fragments together • Now organism is called a Transgenic Organism- organisms that contain functional recombinant DNA (rDNA) from a different organism

  16. III. Uses for Recombinant DNA • 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 factors • Production of insulin • Production of recombinant pharmaceuticals • Plants that produce their own insecticides • Germ line and somatic gene therapy

  17. RECAP • Steps for making a transgenic organism: • Locate and isolate the gene of interest • Cut out the gene and cut the plasmid using the appropriate restriction enzyme

  18. 3. Insert the desired gene into the plasmid matching up the sticky ends

  19. 4. Use the enzyme DNA ligase to seal up the sticky ends

  20. 5. Transfer the vector in the host organism where it will replicate 6. Host organism produces the protein coded for by the recombinant DNA

  21. Insulin Production

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