Genetic Engineering and Recombinant DNA Technology

1. Genetic Engineering and Recombinant DNA Technology • Diversity of life is based on the differences in genes (DNA) • 1 gene = 1 protein = 1 function? • If we change the DNA (genes), then it would be possible to change the function of the cell, and ultimately the organism

2. Genetic Engineering and Recombinant DNA Technology • Recombinant DNA-isolating and combining DNA of one organism with the DNA of a different organism • Human Insulin gene  bacteria • Genetic Engineering-modifying gene(s) to benefit or cure an organism • Agriculture crops, animals • Gene therapy: when cloned genes are used to modify humans, to control genetic diseases  sickle cell, hypercholesterolemia Ice cream anyone?

3. GloFish

4. Cloning Genes • Cloning-production of genetically identical DNA • To clone a gene, must combine human DNA with another organism’s DNA  recombinant DNA • Put gene into a plasmid  extra bacterial chromosome that can accept foreign DNA

5. Molecular Tools Used For Recombinant DNA • How to combine the DNA from 2 different organisms? • DNA must be cut and put back together • What kind of molecule could cut DNA and put it back together again?

6. Molecular Tools Used For Recombinant DNA: Restriction Enzymes • Bacterial enzymes that cut DNA at specific DNA sequences • Leaves single stranded tails at the end of the DNA fragments  ”sticky ends” • DNA fragments cut by same enzyme will come together and form a 2x strand DNA fragment; sticky ends are complementary • DNA ligase closes the “nicks” in the DNA

8. Molecular Tools Used For Recombinant DNA: Polymerase Chain Reaction (PCR) • Multiple copying of a specific DNA sequence Requires: • primers • Heat insensitive DNA polymerase from Thermus aquaticus (Taq) • Used when only have small amounts of DNA and need to do many different tests; genetic identification Cycle 30 = 5.37 x 108

9. Genetic Fingerprinting • Every person has specific DNA sequences that are unique; like fingerprints • During a criminal investigation, those are the sequences that are studied using restriction enzymes, PCR and gel electrophoresis • Gel electrophoresis-isolation of DNA using electrical currents and a gel matrix made of sugars

10. Genetic Fingerprinting: Gel Electrophoresis DNA has (-) charge, it will run towards the bottom of gel that has (+) charge Fragments separate based on size. Match fragment pattern with DNA found at scene. Take DNA found at crime scene Restriction enzymes, STRs, PCR Gel electrophoresis Compare DNA fragmentsname suspect

11. DNA Sequencing • Allows for the actual nucleotide sequence to be known • Automated, done by machines • Used to “solve” all the genes in humans Human Genome Project • Is there an advantage to knowing the entire gene sequence of an organism? • Functional genomics and comparative genomics

12. Applications of Genetic Engineering • In agriculture: • Strawberries that resist frost • rice with beta carotene • tomatoes that last longer • pest resistant corn (95% modified) • Larger animals more meat, milk • In environment: • Bacteria that clean up oil/toxic spills • In medicine: • Insulin, growth hormone, interferon, taxol, relaxin, erythropoietin

13. Genetic Engineering Problems: • Is it safe for human consumption? Allergies? • If organisms get out into natural setting, impact environmental balance? • Bigger animalsmore waste, competition • Hybrids: herbicide resistance gene in corn passed to a weed The “Enviropig” has been genetically modified in such a manner that its urine and feces contain almost 65 percent less phosphorus than usual.

14. Gene Therapy • Introducing the normal gene into humans with disease • We can make the genes through rDNA, but how do we get them inside to every cell? • Ex vivo gene therapy uses modified viruses to get the new gene inside cells  SCID, familial hypercholesterolemia • In vivo gene therapy uses direct injection or inhalation of normal/healthy gene to defective cells/organs • How do we prevent an immune reaction?