Biogenetic Engineering & Manipulating Genes

1. Biogenetic Engineering & Manipulating Genes Chapter 20

2. Intro. Q’s #6-for Chapter 20: Genetic Engineering • What does the acronym PCR stand for and what does this process do? • What does Gel electrophoresis allow us to do? • Give two applications of DNA profiling. • What are the advantages and disadvantages of genetic screening? • Describe what genetic engineering is and explain how such items as restriction enzymes, DNA ligase, and the production of “sticky ends” are used. • Name two “vectors” that can be used for gene transfer. • Give two examples of genetically modified crops or animals • Briefly explain the process of gene therapy and give an example how it works. • Explain what a clone is and how it could be formed. • What are some of the ethical concerns about cloning? Give your opinion if you think cloning is something we shouldbe doing.

3. Genetic Engineering • Chapter 20 • DNA Technology & Genomics

4. O.J. Simpson capital murder case,1/95-9/95 • Odds of blood in Ford Bronco not being R. Goldman’s: • 6.5 billion to 1 • Odds of blood on socks in bedroom not being N. Brown-Simpson’s: • 8.5 billion to 1 • Odds of blood on glove not being from R. Goldman, N. Brown-Simpson, and O.J. Simpson: • 21.5 billion to 1 • Number of people on planet earth: • 6.1 billion • Odds of being struck by lightning in the U.S.: • 2.8 million to 1 • Odds of winning the Illinois Big Game lottery: • 76 million to 1 • Odds of getting killed driving to the gas station to buy a lottery ticket • 4.5 million to 1 • Odds of seeing 3 albino deer at the same time: • 85 million to 1 • Odds of having quintuplets: • 85 million to 1 • Odds of being struck by a meteorite: • 10 trillion to 1

5. Recombinant DNA • Def: DNA in which genes from 2 different sources are linked • Genetic engineering: direct manipulation of genes for practical purposes • Biotechnology: manipulation of organisms or their components to perform practical tasks or provide useful products

6. Tools of Genetic Engineering • Restriction enzymes (endonucleases) -in nature, these enzymes protect bacteria from intruding DNA; they cut up the DNA (restriction); very specific • Restriction site: -recognition sequence for a particular restriction enzyme • Restriction fragments: -segments of DNA cut by restriction enzymes in a reproducible way • Sticky end: -short extensions of restriction fragments • DNA ligase: -enzyme that can join the sticky ends of DNA fragments • Cloning vector: -DNA molecule that can carry foreign DNA into a cell and replicate there (usually bacterial plasmids)

7. Restriction Enzymes (Endonucleases) • Found in Bacteria & named as such: Restriction enzymeName of Bacteria • EcoRI Escherichia coli • Hind III Haemophilus influenza • Pst I Providencia stuartii • Bam I Bacillus amyloliquefaciens • Recognizes Palindromic Sequences • Cuts the all DNA and produces pieces (fragments) called RFLP’s. (Pg. 394) • RFLP: Restriction Fragment length Polymorphisms • RFLP’s can measure genetic relationships and generate a DNA fingerprint when separated on an agarose gel.

8. Gel Electrophoresis Pgs 392-394 • separates nucleic acids or proteins on the basis of size and electrical charge creating DNA bands of the same length • DNA has a net negative charge (use a positive charge in the gel)

9. Introductory Questions #4 • After making the observations from our transformations lab which plate(s) should not have any bacteria growth? Which plate should contain the glowing bacteria under UV light? • Briefly explain the differences between Transformation, Conjugation, and Transduction. How are these three processes the same? (pgs. 348-349) • How is an “F plasmid” different from an “R plasmid”? • What are transposable elements and what do they do? See pg. 351-352. • What do insertion sequences code for? What type of organisms are these sequences found? • How are transposons different from insertion sequences?

10. Introductory Questions #5 • How are repressible operons different from inducible operons? Give an example of each. • What is the difference between an operator and a promoter? • What end of the gel are DNA samples loaded? Do the larger or shorter fragments move farther in the gel? Explain your answer. • What does the acronym PCR stand for and what does this process do? • What does Gel electrophoresis allow us to do? • Give two applications of DNA profiling.

11. Loading the Gels w/Micropipette • Be sure that your Micropipette is set at “10.0” • Load your DNA samples into the correct well as follows from left to right using a micropipette. Closet to the corner cut of the gel. • Well #1: Lambda DNA (uncut) (yellow tube) • Well #2: Lambda cut w/PstI (blue tube) • Well #3: Lambda cut w/EcoRI (pink tube) • Well #4: Lambda cut w/Hind III (green tube) • Once all four wells are filled, carefully bring your chamber to the front or back and connect the leads: Black “-” Red “+” While you wait read and answer all the questions from your guide sheet. Pgs. 21-28

13. Seven Restriction sites (8 fragments) using Hind III Pg. 29

14. Restriction Enzymes used in the Lab Name of EnzymeSequence Recognized “Palindrome” -EcoRI G ….A A T T C C T T A A…. G -PstI C T G C A G G A C G T C -HindIII A A G C T T T T C G A A

15. Restriction Enzymes

16. Measure (mm) Visible Fragments  DNA  DNA  DNA  DNA Uncut w/PstI w/EcoRI HindIII Fragment #1 Fragment #2 Fragment #3

17. Setting up a Gel Electrophoresis Unit

18. Gel Electrophoresis • DNA fragments placed into “wells” in gel agarose • Electricity pulls on DNA fragments • Fragments travel at different rates based on size and ability to squeeze through swiss-cheese-like agarose

19. Action of Restriction Enzymes-Pg. 386

20. Restriction Fragment Analysis • Restriction fragment length polymorphisms (RFLPs) • Southern blotting: process that reveals sequences and the RFLPs in a DNA sequence • DNA Fingerprinting (DNA Profiling)

21. Producing Restriction Fragments • DNA ligase used to splice together cut plasmids and chromosome fragments

22. DNA Libraries • Collection of DNA fragments that have been incorporated into plasmids

23. Steps for Eukaryotic Gene Cloning • Isolation of cloning vector (bacterial plasmid) & gene-source DNA (gene of interest) • Insertion of gene-source DNA into the cloning vector using the same restriction enzyme; bind the fragmented DNA with DNA ligase • Introduction of cloning vector into cells (transformation by bacterial cells) • Cloning of cells (and foreign genes) • Identification of cell clones carrying the gene of interest

24. Tools for DNA Analysis & Genomics • PCR (polymerase chain reaction) • Gel electrophoresis • Restriction fragment analysis (RFLPs) • Southern blotting • DNA sequencing • Human genomeproject

25. Polymerase Chain Reaction (PCR)http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html# • Amplification of any piece of DNA without cells (in vitro) • Produces many identical copies of a DNA segment • Materials: heat, DNA polymerase, nucleotides, single-stranded DNA primers • Applications: fossils, forensics, prenatal diagnosis, etc.

26. Polymerase Chain reaction (PCR)

27. Polymerase Chain Reaction • PCR = common method of creating copies of specific fragments of DNA • PCR rapidly amplifies a single DNA molecule into many billions of molecules. • Small samples of DNA can produce sufficient copies to carry out forensic tests

28. DNA Profiling (DNA fingerprinting) Several Applications: -Used in criminal investigations -Identify the remains of dead people -Paternity Tests -Screening for genetic disorders

29. Polymerase Chain Reaction • PCR = common method of creating copies of specific fragments of DNA • PCR rapidly amplifies a single DNA molecule into many billions of molecules. • Small samples of DNA can produce sufficient copies to carry out forensic tests

30. DNA Sequencing • Determination of nucleotide sequences (Sanger method, sequencing machine) • Genomics: the study of genomes based on DNA sequences • Human Genome Project

31. Practical DNA Technology Uses • Diagnosis of disease • Human gene therapy • Pharmaceutical products (vaccines) • Forensics • Animal husbandry (transgenic organisms) • Genetic engineering in plants • Ethical concerns?

32. Genetic Screening Def: Testing individuals in a population for the presence or absence of a gene (allele) Advantages: -pre-natal diagnosis of genetic disorders -Could help stop the spread of a disorder -Can detect carriers of a potential disorder Disadvantages: -invasion of privacy -Individuals can become stigmatized in the community -Discriminated against or feared -Employment and medical insurance

33. Cloning

34. Bacterial plasmids in gene cloning

35. Steps for Eukaryotic gene cloning

36. Assignment Packet #4 • Study Guide • Pre-Lab Bacteria Transformation w/Glo • Pre-Lab Analysis of Lambda DNA • Videos x2 • IQ’s x5 • Cumulative test Ch. 16-20

37. Plant Cloning • Tissue Culture Propagation • Bits of phloem can be induced in the lab to form clumps of tissue that will make roots & shoots • Orchid culture

38. Embryo Cloning • Medical technique which produces identical twins or triplets • Duplicates nature • One or more cells are removed from a fertilized embryo, encouraged to develop into one identical twins or triplets • Done for many years on animals • Limited experimentation on humans

39. Adult DNA Cloning • Cell nuclear replacement • Produces a duplicate of an existing animal • DNA from an embryo is removed; • Replaced with DNA from an adult animal • Embryo is implanted in a womb and allowed to develop into a new animal • Untried on humans - potential of producing a twin of an existing person

40. Therapeutic Cloning • Stem cells removed from an embryo with intent of producing tissue or a whole organ for transplant back into the person who supplied the new DNA • Embryo dies in the process • Goal is to produce a healthy copy of a sick person's tissue or organ for transplant

41. Therapeutic Cloning • Vastly superior to organ transplants • Supply would be unlimited - no waiting lists • Tissue or organ would have the sick person's original DNA • No immunosuppressant drugs would need to be taken

42. DNA Cloning • Restriction enzymes (endonucleases) -in nature, these enzymes protect bacteria from intruding DNA; they cut up the DNA (restriction); very specific • Restriction site: -recognition sequence for a particular restriction enzyme • Restriction fragments: -segments of DNA cut by restriction enzymes in a reproducable way • Sticky end: -short extensions of restriction fragments • DNA ligase: -enzyme that can join the sticky ends of DNA fragments • Cloning vector: -DNA molecule that can carry foreign DNA into a cell and replicate there (usually bacterial plasmids)

43. Adult DNA Cloning

44. Adult DNA Cloning

45. Stem cell clon-ing

47. Theraputic Stem Cell Cloning • Used

48. Intro. Q’s for Chapter 14: Genetic Engineering • What does the acronym PCR stand for and what does this process do? • What does Gel electrophoresis allow us to do? • Give two applications of DNA profiling. • What are the advantages and disadvantages of genetic screening? • Describe what genetic engineering is and explain how such items as restriction enzymes, DNA ligase, and the production of “sticky ends” are used. • Name two “vectors” that can be used for gene transfer. • Give two examples of genetically modified crops or animals • Briefly explain the process of gene therapy and give an example how it works. • Explain what a clone is and how it could be formed. • What are some of the ethical concerns about cloning? Give your opinion if you think cloning is something we shouldbe doing.

49. Chromatin • Def: complex of DNA and proteins • DNA Packing • Histone proteins (+ charged amino acids w/ phosphates of DNA that are - charged) • Nucleosome -”beads on a string”; basic unit of DNA packing • Heterochromatin -highly condensed interphase DNA (can not be transcribed) • Euchromatin -less compacted interphase DNA (can be transcribed)

50. Def: Unit of genetic function consisting of coordinately related clusters of genes with related functions (transcription unit) Operons, II • Inducible (lac operon): • lactose metabolism • lactose not present: repressor active, operon off; no transcription for lactose enzymes • lactose present: repressor inactive, operon on; inducer molecule inactivates protein repressor (allolactose) • transcription is stimulated when inducer binds to a regulatory protein