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DNA/GENE TECHNOLOGY

DNA/GENE TECHNOLOGY. Chapter 9. DNA fingerprinting. The promise and perhaps perils of embryonic stem cells. Types of Genetic Engineering. Selective Breeding GMO’s Gene Sequencing Gene Cloning/Pharmaceutical Production DNA Fingerprinting Transgenic Organisms

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DNA/GENE TECHNOLOGY

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  1. DNA/GENE TECHNOLOGY Chapter 9

  2. DNA fingerprinting The promise and perhaps perils of embryonic stem cells Types of Genetic Engineering • Selective Breeding • GMO’s • Gene Sequencing • Gene Cloning/Pharmaceutical Production • DNA Fingerprinting • Transgenic Organisms • Therapeutic Cloning/Stem Cells • Reproductive Cloning • Gene Therapy • Human Genome Project

  3. Engineering vs. Technology • Genetic Engineering • The process and outcome of making changes in the DNA code of living organisms • Genetic Technology • The tools and instruments used and developed for the process of manipulating genes

  4. Selective breeding • Produces organisms with desired traits • 2 Types – Inbreeding & Hybrids • What traits might breeders want to select for in these organisms?

  5. Inbreeding • Mating of closely related individuals • Ensures that offspring remain homozygous for most traits • Keeps wanted traits in the breed • Keeps out un-wanted traits Pure-Bred (inbred) Bulldog Inbred Weimeraner Dog

  6. Inbreeding • Does have a high risk of offspring receiving 2 HARMFUL recessive alleles that were present in the family line • Which means??? • Causes mutations that ALREADY exist to pair at a higher frequency

  7. Hybrids • Usually produce larger, stronger organisms • AND VARIATION! • Mixing dominant & recessive keeps out harmful recessive phenotypes • ESPECIALLY IN PLANTS!

  8. HybridsDogs and Ligers and Geep – oh my! Hybridization – Mating of slightly dissimilar organisms to produce desired combination Must have same chromosome number, similar structure Liger Geep – Hybrid of Goat and Sheep

  9. Genetically Modified Organisms: GMOs • Altering the genetics of plants or animals for human benefit • One of the first was Bt cotton: • Bacterial gene from Bacillus thuringiensis was put in cotton • Made it toxic to insects • Specifically the Boll Weevil GMO Cotton (contains a bacterial gene for pest resistance); 80% of all cotton Standard Cotton

  10. Genetically Modified OrganismsGMOs • Altering the genetics of plants and animals for human consumption • Polyploidy – chemicals disrupt meiosis – bigger fruit or seedless fruit

  11. Fourteen month-old genetically engineered (GMO) salmon (left) and standard salmon (right). Genetically Modified OrganismsGMOs

  12. Transgenic organisms • Organisms with genes from other species • Ex: Mice with jellyfish gene – will glow!!! • Benefits of transgenics: • Gene function determination • Medical studies, drug trials • Creation of medical proteins/drugs • Hybrid organisms/GMO’s • EX: Human chemotherapy drug in chickens

  13. Transgenic vs. Clone Transgenic Organisms have genes inserted from another organism Cloned Organisms have the exact same DNA as another organism Tobacco Plant with firefly gene Dolly the cloned sheep

  14. Cloning

  15. Types of cloning • Gene cloning • Inserting just one gene into org to copy • Therapeutic cloning • Stem cells  new cells/organs • Reproductive cloning • Creating an entire organism

  16. Therapeutic Cloning/Stem Cells • Stem cells – undifferentiated cells • Can develop into any type of cell in body • Embryonic* – most potential for success • But regulated by law • Bone marrow/Spinal – some potential for success

  17. Possible clone use… • Cloning new stem cells to repair tissues

  18. Stem Cells are Found in the Adult, but the Most Promising Types of Stem Cells for Therapy are Embryonic Stem Cells

  19. Therapeutic Cloning/Stem Cells Therapeutic cloning- cloning of specific cells/tissues/organs; not whole organism

  20. Some Thorny Ethical Questions Are these masses of cells a human? Is it ethical to harvest embryonic stem cells from the “extra” embryos created during in vitro fertilization?

  21. Gene Cloning • Cloning a single GENE in an organism… • Organism copies gene through replication • Produces proteins

  22. Reproductive Cloning • Making an identical organisms genetically speaking • Steps: • 1. Take DNA (nucleus) from existing org. • 2. Take egg cell and replace its nucleus (DNA) with existing org. DNA (nucleus) • 3. Allow egg cell to develop into offspring • 4. Offspring will have same DNA as existing organism • Why should we clone? • Food industry? • Endangered species? • Problems/Ethics? Dolly I and Dolly II – her clone

  23. (Science (2002) 295:1443) Reproductive Cloning - Pet Cloning? University of Texas 2002 – Success Rate of 1/87 embryos Significantly, Carbon Copy is not a phenotypic “carbon copy” of the animal she was cloned from Environmental factors and proteome interactions cause phenotypic differences Nature vs. Nurture argument FYI – cost $ 3.7 million

  24. Clones

  25. Possible clone use… • Clone successful plants

  26. Gene Therapy • Vectors-carry DNA from one source to another. Useful in gene therapy and making recombinant DNA • A virus is often used • Knock out the viral DNA and add desired gene to ‘infect’ patient

  27. Gene Therapy • Restriction enzymes cut out ‘normal’ gene from genome sample • Take out viral DNA and add ‘normal’ human gene to virus • Viral vector infects patient with ‘normal’ gene to replace mutated one • Normal gene inserts into patient’s DNA and now produces proper protein/trait • Ex: normal CF gene being infected into a cystic fibrosis patient

  28. Creating Recombinant DNA 1. Cut (cleave) DNA from one organism with a restriction enzyme 2. Insert (splice) the wanted genes (DNA) from another organism (Fig 13.4) • RESULT: • RECOMBINANT DNA = TRANSGENIC ORGANISM • Usually done on plasmid DNA = (bacterial) circular DNA

  29. What if there isn’t enough DNA in the sample? • Tiny amounts of DNA can be amplified by a technique called PCR (polymerase chain reaction)

  30. PCRMaking enough DNA to read • Three – step amplification cycle • Cycles of heating and cooling • Causes DNA to separate (DENATURE) and then come back together (ANNEAL) • Use DNA Polymerase • Generates MORE DNA a certain size of DNA fragment (from one sample) PCR

  31. Now that we have enough DNA… What’s next?

  32. CUTTING DNARestriction Enzyme We will use TA-ase, an imaginary enyzme, to cut our DNA Sample DNA strand CTGGCTAGGCTACCATGCCCGTAAAT Restriction Enzymes

  33. CUTTING THE DNARestriction Enzyme We will use TA-ase, an imaginary enyzme, to cut our DNA Sample DNA strand CTGGCTAGGCTACCATGCCCGTAAAT CTGGCTA GGCTA CCATGCCCGTA AAT

  34. SEPARATING THE DNAGel Electrophoresis Electricity separates fragments by size in a gel Largest fragment travels least Smallest the most Gel Electrophoresis

  35. DNA is slightly (-), thus it will move towards (+)

  36. Here are our DNA fragments Which one will travel fast and far? WHY? Which one will travel slow and short? WHY? CTGGCTA GGCTA CCATGCCCGTA AAT 1 3 4 2

  37. SEPARATING THE DNAGel Electrophoresis CCATGCCCGTA CTGGCTA GGCTA AAT

  38. SEPARATING THE DNA Gel ElectrophoresisRESULTS…DNA “Fingerprint” • Can be used to ID persons • Very effective means of: • Criminal identification & exclusion • Paternity cases • Missing persons • Entire DNA is not used, only portions known to differ from individual to individual • Gel is sometimes called an “autoradiograph” or “autorad” M = Marker (control) DNA

  39. DNA FINGERPRINT:THE LAB BASICS (A SUMMARY) • PCR = to increase the amount of DNA • Restriction enzymes = to cut the DNA into different sized fragments • Gel Electrophoresis = to separate fragments according to size • CONCLUSION = Try to match fragments from different samples

  40. STEP 4 = READINGHow do you read a DNA fingerprint? Victim’s DNA finger print

  41. STEP 4 = READINGHow do you read a DNA fingerprint? A B C D E Victim’s DNA finger print Which sample is a match?,

  42. STEP 4 = READINGHow do you read a DNA fingerprint? A B C D E Victim’s DNA finger print Which sample is a match?,

  43. STEP 4 = READINGHow do you read a DNA fingerprint? C Victim’s DNA finger print Which sample is a match?,

  44. ANALYZING DNA SAMPLES • Let’s try some…

  45. DNA FINGERPRINTING • Comparing different samples of DNA

  46. Paternity Testing • Not just matching evidence to suspect…

  47. Gene Sequencing – How do we know what a gene does? • Gene cloning (in a bacterium) protein synthesis  analysis of amino acid sequence • Gene “knockout” in “lab” animals • Comparative sequences in people with disease/without

  48. Gene Sequencing – How do we know what the DNA is? • Take gene cut by restriction enzymes • Put gene into bacteria/rat to see what protein does • Use florescent DNA probes to bind to complementary sequences • Ex: Glowing ACT DNA probe would attach to TGA DNA & id that sequence

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