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DNA TECHNOLOGY

DNA TECHNOLOGY. Genetic engineering and all that stuff. Glowing mice. An overview of how bacterial plasmids are used to clone genes. Tools and Techniques Part 1. Restriction Enzymes DNA Ligase Cloning Vectors Host Organisms Cloning of DNA/genes. Restriction Endonucleases. Eco RI

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DNA TECHNOLOGY

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  1. DNA TECHNOLOGY Genetic engineering and all that stuff

  2. Glowing mice

  3. An overview of how bacterial plasmids are used to clone genes

  4. Tools and Techniques Part 1 • Restriction Enzymes • DNA Ligase • Cloning Vectors • Host Organisms • Cloning of DNA/genes

  5. Restriction Endonucleases • Eco RI --GAATTC-- --G AATTC-- --CTTAAG-- --CTTAA G-- • SmaI --CCCGGG-- --CCC GGG-- --GGGCCC-- --GGG CCC-- Recognition sequenceResults of cutting

  6. Using a restriction enzyme and DNA ligase to make recombinant DNA

  7. Restriction enzymes movie Video Link

  8. Tools and Techniques Part 1 • Restriction Enzymes • DNA Ligase • Cloning Vectors • Host Organisms • Cloning of DNA/genes

  9. pUC19 A plasmid “cloning vector” pUC19 with Insert: Note the insert disrupts the gene coding for b-galactosidase.

  10. Tools and Techniques Part 1 • Restriction Enzymes • DNA Ligase • Cloning Vectors • Host Organisms • Cloning of DNA/genes

  11. Cloning a gene: The Movie Video Link

  12. Cloning a human gene in a bacterial plasmid: a closer look

  13. Cloning a human gene in a bacterial plasmid: a closer look

  14. Cloning a human gene in a bacterial plasmid: a closer look

  15. Tools and Techniques Part 2 • Gel Electrophoresis • DNA synthesis • DNA sequencing • Reverse transcriptase • RFLP Analysis • PCR (Polymerase Chain Reaction)

  16. Gel Electrophoresis of Macromolecules

  17. Gel Electrophoresis

  18. Tools and Techniques Part 2 • Gel Electrophoresis • DNA synthesis • DNA sequencing • Reverse transcriptase • RFLP Analysis • PCR (Polymerase Chain Reaction)

  19. DNA sequencers

  20. Tools and Techniques Part 2 • Gel Electrophoresis • DNA synthesis • DNA sequencing • Reverse transcriptase • RFLP Analysis • PCR (Polymerase Chain Reaction)

  21. Making complementary DNA (cDNA) for a eukaryotic gene

  22. Tools and Techniques Part 2 • Gel Electrophoresis • DNA synthesis • DNA sequencing • Reverse transcriptase • RFLP Analysis • PCR (Polymerase Chain Reaction)

  23.   Using restriction fragment patterns to distinguish DNA from different alleles

  24. Laboratory worker reviewing DNA band pattern

  25. Tools and Techniques Part 2 • Gel Electrophoresis • DNA synthesis • DNA sequencing • Reverse transcriptase • RFLP Analysis • PCR (Polymerase Chain Reaction)

  26. PCR (Polymerase Chain Reaction) • Allows any piece of DNA to be rapidly amplified ! • Only minute amounts of DNA are needed ! PCR animation link #1

  27. PCR procedure • Heat to ~ 95 degrees to denature the DNA strands (Strands separate) • Cool and use the separated DNA strands as templates using a heat tolerant DNA Polymerase (e.g. Taq polymerase from Thermus aquaticus) • Repeat cycle … Repeat cycle …Repeat cycle … Repeat cycle … Repeat cycle …

  28. The polymerase chain reaction (PCR) PCR animation link #2

  29. Human Genome Project

  30. Applications • Diagnosis of Disease • Gene Therapy • Vaccines and other Pharmaceuticals • Forensic use • Environmental use (bioremediation) • Agricultural use * Transgenic animals * Transgenic plants

  31. One type of gene therapy procedure

  32. Gene Therapy for skin cancer • Mark Origer, 53, After five years of losing the battle with the disease, he was made well enough to attend his daughter's wedding.

  33. Genetic Testing

  34. Genetic Testing (NPR story) Reading Genes for Disease, Part 4: Dilemmas http://www.npr.org/templates/story/story.php?storyId=1915471 Ability to Cure Disease Lags Behind Ability to Predict It. All Things Considered,May 30, 2004 · Tests that can reveal a person's risk of a disease are an advance of modern medicine. But they are also perceived as a double-edged sword: The ability to diagnose a disease or to predict its arrival has outstripped the ability to treat it.

  35. Some Currently Available DNA-Based Gene Tests Alpha-1-antitrypsin deficiency (AAT; emphysema and liver disease) Amyotrophic lateral sclerosis (ALS; Lou Gehrig's Disease; progressive motor function loss leading to paralysis and death) Alzheimer's disease* (APOE; late-onset variety of senile dementia) Ataxia telangiectasia (AT; progressive brain disorder resulting in loss of muscle control and cancers) Gaucher disease (GD; enlarged liver and spleen, bone degeneration) Inherited breast and ovarian cancer* (BRCA 1 and 2; early-onset tumors of breasts and ovaries) Hereditary nonpolyposis colon cancer* (CA; early-onset tumors of colon and sometimes other organs) Charcot-Marie-Tooth (CMT; loss of feeling in ends of limbs) Congenital adrenal hyperplasia (CAH; hormone deficiency; ambiguous genitalia and male pseudohermaphroditism) Cystic fibrosis (CF; disease of lung and pancreas resulting in thick mucous accumulations and chronic infections) Duchenne muscular dystrophy/Becker muscular dystrophy (DMD; severe to mild muscle wasting, deterioration, weakness) Dystonia (DYT; muscle rigidity, repetitive twisting movements) Fanconi anemia, group C (FA; anemia, leukemia, skeletal deformities) Factor V-Leiden (FVL; blood-clotting disorder) Fragile X syndrome (FRAX; leading cause of inherited mental retardation) Hemophilia A and B (HEMA and HEMB; bleeding disorders) Hereditary Hemochromatosis (HFE; excess iron storage disorder) Huntington's disease (HD; usually midlife onset; progressive, lethal, degenerative neurological disease) Myotonic dystrophy (MD; progressive muscle weakness; most common form of adult muscular dystrophy) Neurofibromatosis type 1 (NF1; multiple benign nervous system tumors that can be disfiguring; cancers) Phenylketonuria (PKU; progressive mental retardation due to missing enzyme; correctable by diet) Adult Polycystic Kidney Disease (APKD; kidney failure and liver disease) Prader Willi/Angelman syndromes (PW/A; decreased motor skills, cognitive impairment, early death) Sickle cell disease (SS; blood cell disorder; chronic pain and infections) Spinocerebellar ataxia, type 1 (SCA1; involuntary muscle movements, reflex disorders, explosive speech) Spinal muscular atrophy (SMA; severe, usually lethal progressive muscle-wasting disorder in children) Thalassemias (THAL; anemias - reduced red blood cell levels) Tay-Sachs Disease (TS; fatal neurological disease of early childhood; seizures, paralysis) Over 900 tests are currently available

  36. PHARM animals Transgenic Sheep Carrying a human gene for a human protein which they secrete in their milk. The protein is then purified from their milk

  37. Transgenic organisms

  38. Forensic use of DNA patterns“DNA Fingerprints”

  39. DNA fingerprints from a murder case

  40. DNA Forensics (movie)

  41. DNA evidence is now readily accepted in courts. In addition to convicting perpetrators of crimes it has also been used to exonerate the innocent. The Bernard Webster case http://www.innocenceproject.org/Content/290.php

  42. “Golden” rice contrasted with ordinary rice Transgenic Golden Rice (High levels of Beta-carotene –used to make Vitamin A – give the rice a golden color.

  43. Using the Ti plasmid as a vector for genetic engineering in plants TRANSGENIC PLANT

  44. Safety and Ethics GMO (genetically modified organism) food controversy

  45. Scare tactics?

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