Biogenetic Engineering & Manipulating Genes

1. Biogenetic Engineering & Manipulating Genes Chapter 20

2. Intro. Q’s #5 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? • How is a cDNA library different from a genomic library? (p. 388-390) • Name two “vectors” that can be used for gene transfer. • Give two examples of a genetically modified crop or animal. • 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 Pgs. 384-396 & 402-408

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. Introduction Manipulating genes and sequencing DNA has become one of the greatest achievements in the last 50 years -First complete genome sequenced was a bacteria (1995) -Human Genome project (1990-2003)

6. 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

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

8. 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 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)

9. Restriction Enzymes (endonucleases)http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#

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

11. Cloning

12. Steps for Eukaryotic Gene Cloning (pg. 387) • 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 • http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#

13. DNA Libraries (pg. 388) • Collection of DNA fragments that have been incorporated into plasmids

14. Genomic Libraries (pg. 388) • Stored amount of cloned genes within bacteria or virues (bacteriophages) • Contained as cloned plasmids within bacteria or as fragments within Bacteriophages genome • Phages can carry a larger DNA insert vs. bacteria

15. cDNA Library (Pg. 390) • cDNA (complementary DNA) • Start w/mRNA to make a ssDNA strand • Use an enzyme “reverse transcriptase” which is found in retroviruses such as HIV (see pg. 340-341) to make a ssDNA strand. • A complementary strand of DNA is made by DNA polymerase to produce a dsDNA molecule (cDNA) http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#

16. 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.

17. 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

18. Polymerase Chain reaction (PCR)

19. Inserting DNA into the nucleus of a cell

20. Gel Electrophoresis (Lab) • 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

21. 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)

22. Gel Electrophoresis • 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)

23. Southern Blot of DNA Fragments (pg. 395) • Used to determine the presence or absence of a particular nucleotide sequence in the DNA different sources. • Compare the restriction fragments produced from the different samples. • Radioactive probes are used and attach to the DNA fragments by complementary base pairing. http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#

24. DNA Profiling (DNA fingerprinting) Applications: -Used in criminal investigations -Identify the remains of dead people -Compare Evolutionary origins of organisms -Look for genetic disorders (mutations) -Sequence DNA samples

25. DNA Fingerprinting

26. Video #3: Using DNA evidence to Solve Crimes • Write 15 key statements from the video. • Please number each statement. • Indicate where the sources of DNA were found for each case.

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

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

29. 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

30. Bacterial plasmids in gene cloning

31. Steps for Eukaryotic gene cloning

32. 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

33. 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

34. 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

35. 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

36. Application: Gene Therapy

37. 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

38. Adult DNA Cloning

39. Adult DNA Cloning

40. Stem cell clon-ing

42. Theraputic Stem Cell Cloning • Used

43. 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)