Understanding DNA Technology: From Restriction Enzymes to Recombinant DNA

1. Figure 12.12B Honors Biology: DNA Technology and Society

2. 14.3 Manipulating DNA • Cutting, Separating and Reading DNA • Restriction Enzymes • Gel Electrophoresis • Probes

3. Enzymes are essential tools in DNA technology 0 Restriction enzymes (endonucleases) • Made by bacteria to cut out foreign DNA • cut DNA at specific sequences – used like molecular scissors • Recognition sequences: 4 to 6 bp’s long • Some cut and leave “sticky ends” • Bacteria methylate A’s and C’s to protect own DNA from being cut up • Ex: EcoR1 DNA ligase • used to “paste” DNA fragments together Restriction enzyme animation

4. RESTRICTION ENZYMES aka Molecular Scissors

5. 0 Gel electrophoresis sorts DNA fragments by size • a molecular sieve (jello) to separate chunks of DNA based on size • restriction enzymes used to chop up DNA into RFLP’s • RFLP: restriction fragment length polymorphism • process utilizes negative charge of DNA to move pieces thru the gel • bigger pieces stay close to origin, smaller pieces move farther toward the positive end • result is a DNA fingerprint (bar code) of your specific DNA pieces…everyone’s DNA will chop up differently fingerprint is unique RFLP animation

7. The Human Genome Project : a major application of DNA technology 0 • Began in 1990: involved genetic and physical mapping of chromosomes and DNA sequencing • Data provide insight into development, evolution, and diseases • Most of the human genome does not consist of genes • The haploid human genome contains about 25,000 genes and a huge amount of noncoding DNA • noncoding DNA: repetitive nucleotide sequences (“junk DNA”) and transposons that can move about within the genome • repetitive sections found at centromere and at tips of chromosomes (telomeres) provide chromosome structure • * telomeres have protective function for chromosomes • * significant loss of telomeric DNA quickly leads to cell death. • * abnormally long telomeres are linked to cancer cell immortality

8. The science of genomics compares whole genomes 0 • The sequencing of many prokaryotic and eukaryotic genomes • Nonhuman genomes can be compared with the human genome

9. http://www.tmz.com/2014/03/06/michael-jackson-son-brandon-howard-dna-test/http://www.tmz.com/2014/03/06/michael-jackson-son-brandon-howard-dna-test/

10. Chapter 15 Genetic Engineering • 15.1 – Selective Breeding • Selective Breeding • Hybridization • Inbreeding • Biotechnology • Bacterial Mutations • Polyploid Plants

11. Selective Breeding: Hybridization and Inbreeding Selective Breeding: takes advantage of naturally occurring variations and passes them to next generation ex) corn has been highly selected by native Americans for centuries and changed from a useless grass to the most productive food crop on the planet. 1. Hybridization: crossing of dissimilar individuals to get the best of both into the offspring ex: disease resistance of one plus the crop yield of the other 2. Inbreeding: the continued breeding of those with similar characteristics ex: dog breeds are inbred to keep gene pool constant for those particular traits unique to that “breed” **down side: because all are so similar, you increase the chance that 2 recessive alleles for a disease join. Now that disease stays in that gene pool and is tuned over in a high frequency. (ex: hip problems in labs, arthritis in golden retreivers)

12. Q: How can we increase variation in a species? A: Cause Mutations 1. Radiation and chemical exposure of bacteria * most mutations are harmful, but a few prove beneficial for a particular environment ex: oil-digesting bacteria ex: attempts to mutate bacteria to “eat” radioactive waste and render it stable ex: attempts to mutate bacteria to digest metals and clean the environment of industrial waste • Polyploidism (in plants) * use chemicals that don’t allow chromosomes to separate during meiosis get a 2N egg or 2N pollen (sperm) * result? 3N or 4N plant * new polyploid species are bigger and stronger than diploid relatives * ex: bananas and other vital crops

13. 15.2 Recombinant DNA • Southern Blot • PCR • Recombinant DNA • Plasmids • Transformation • Genetic Marker • Transgenic Organisms • Clone

14. PCR is used to amplify DNA sequences 0 (PCR)polymerase chain reaction • used to clone a small sample of DNA quickly • produces enough copies for analysis • used when DNA source is scant or impure • in a few hours, PCR yields 100 Billion copies of one gene PCRanimation Figure 12.14

15. Changing DNA • Early work = Griffith’s experiments on bacterial transformation (recall from chapter 10) • A cell takes in DNA from outside the cell and becomes a part of that organism’s genome

16. BACTERIAL PLASMIDS AND GENE CLONING 0 Plasmids are used to customize bacteria • Plasmids are extra rings of DNA outside the bacterial nucleoid • Researchers can insert desired genes into plasmids, creating recombinant DNA plasmids (rDNA) • The new plasmids are inserted into other bacteria • If the recombinant bacteria multiply into a clone, the foreign genes are also copied • The bacteria can also express the new gene and make the protein • Ex: insulin production real plasmid • Bacteria are used as: • copy machines (to clone genes) • factories (to make protein of inserted gene)

17. 0 Bas i c Process Figure 12.1

18. Plasmid DNA Transformation Fig. 15.10

19. 0 Cloning a gene in a bacterial plasmid • Sometimes a genetic marker is used to ‘see’ if the bacteria has accepted the new DNA (a gene that is resistant to antibiotics, one that glows, etc.) Cloning animation Figure 12.3

20. Transgenic Organisms Transgenic – containing genes from other species Can be produced by insertion of recombinant DNA into the genome of host organism Transforming a Plant Cell Possible bc of universal genetic code Can increase food supply

21. Transgenic Animals: contain genes from other animals 0 • Genes from other organisms are inserted into their genomes • Involves in vitro fertilization and injection of desired gene directly into fertilized eggs • Engineered embryos are implanted into a surrogate mother • Ex: pigs with human cell lines for organ donation • Ex: chickens produce eggs with additional proteins Q : Is it ethical? What are the risks? What happens when a GM crops pass genes for pesticide and herbicide resistance to weeds??  superweeds that would be very difficult to destroy

22. Examples of Transgenic Organisms

23. To Clone or Not to Clone? 0 • A clone is an individual created by asexual reproduction • genetically identical to a single parent • Cloning has many benefits but evokes just as many concerns

24. Nuclear transplantation is used to clone animals 0 • * Reproductive cloning of nonhuman mammals is useful in research, agriculture, and medicine • * Therapeutic cloning produces stem cells which can perpetuate themselves in culture and give rise to specialized cells cloning stem cell research

25. Yes, the jokes are FREE!!!!!

26. 15.3 Applications of Genetic Engineering • Health and Medicine • Gene Therapy • DNA Microarray • DNA Fingerprinting • Forensics

27. GENETICALLY MODIFIED (GM) ORGANISMS • Recombinant DNA technology is producing new genetic varieties of plants and animals • Use Ti plasmid of Agrobacterium tumefaciens as the vector GM plant • ex: soybeans and cotton crops receive bacterial genes to make them resistant to herbicides and pests • ex: “golden rice” = rice with a few daffodil genes added. Rice plant can now make B-carotene, needed for vitamin A production in humans. Vitamin A deficiency (and resulting blindness) is a serious problem for ½ of the world who depend on rice as their staple food. Ti plasmid animation

28. Recombinant cells and organisms can mass-produce gene products for medicinal and other purposes 0 1982: Humulin The first recombinant drug made by bacteria and approved by the FDA

29. Mass-Produced Gene Products cont’d • Bacteria with plasmid: get gene product in large quantity ex: insulin • S. cerevisiae yeast: eukaryotic cell with plasmids can produce eukaryotic proteins better ex: proteins for hepatitis B vaccine • Mammalian cells: can process large proteins better ex: Factor 8 (fight hemophilia), TPA (fight heart attacks) and EPO (fight anemia) • Whole organism: gene is added to genome and the gene product (protein) is then produced in the organism ex: human gene into cows to make milk with human protein ex: human gene into sheep to make milk with a blood protein to fight CF HepB vaccine animation

30. Gene Therapy 0 • Is the alteration of an afflicted individual’s genes • Use a harmless recombinant virus as a vector (deliverer of needed gene) • Remove bone marrow cells and treat with recombinant virus • “infected” cells with injected gene are put back into patient. • Patient now has needed gene in bone marrow cells • May one day be used to treat both genetic diseases and non-genetic disorders. Unfortunately, progress is slow Figure 12.13

31. Technique used to study LOTS of genes at once and to understand their activity levels ssDNA spots are attached to a glass slide (spots contain different fragments) Colored tags are used to label the source of DNA EX. compare cancer genes with normal genes DNA Microarray

32. 0 1 2 RFLP’s used to detect differences in DNA sequences • Used in crime scene investigations to show guilt or innocence of suspect • Body fluids left behind are processed and analyzed through gel electrophoresis

33. DNA Probes Can Detect Harmful Alleles 0 • Radioactive probes can reveal DNA bands of interest on a gel • Used in genetic screening tests • Huntington’s Disease • Cystic Fibrosis #3. DNA bands treated to separate double strands. Single strands blotted off onto filter paper. #4. Blotted paper is treated with radioactive probe (complimentary to gene sequence of disease causing gene) Probe attaches to RFLP’s from original gene …get several bands #5. Unattached probe rinsed off. Photographic film placed on blot paper. Radioactivity exposes film, forms image corresponding to DNA which base-paired with probe person I has Huntington’s. Persons II and III are being tested…results?

34. Crime Scenes and DNA Evidence Investigator at one of the crime scenes (above), Narborough, England (left) 0 • Many violent crimes go unsolved for lack of enough evidence • If biological fluids are left at a crime scene, DNA can be isolated from them • DNA fingerprinting determines with near certainty whether two samples of DNA are from the same individual

35. Blood from defendant’s clothes Defendant’s blood Victim’s blood Figure 12.12B 0 DNA fingerprinting can help solve crimes, paternity suits Figure 12.12A Q: Did he do it? Fingerprint (12E) activity and Discovery channel “Forensics” video

36. The Simpsons’ Background information: Homer got into a dispute at a local establishment. To avoid a standoff, Homer takes his family to his father’s farm to hide out. We join Homer and his family as they arrive at the farm. TOMACCO Explain, in detail, how this Simpsons’ clip relates to genetic engineering.

37. Southern Blot • Technique for finding specific DNA sequencesusing a labeled piece of nucleic acid as a probe