Pan, Cerberus, and the Centaurs: Genetic Engineering

1. Pan, Cerberus, and the Centaurs: Genetic Engineering Donna C. Sullivan, PhD Division of Infectious Diseases University of Mississippi Medical Center

2. Fun Things To Do With DNA • Spool it onto a glass rod (Isolation) • Put it in a necklace (Precipitation) • Pull it apart (Denature) and put it back together (Anneal) • Cut it up (Restriction Enzymes) and look at it (Gel electrophoresis) • Map it (Southern Blots) • Read it (Sequencing) • Copy it millions of times (Cloning, Polymerase Chain Reaction) • Move it from one place to another (Genetic Engineering)

3. Diversity of Genetic Material • Prokaryotic DNA • Eukaryotic DNA • Viral DNA or RNA • Plasmid DNA

4. Enzymatic or Chemical Extraction Cell Lysis Isolating Nucleic Acids forMolecular Analysis

5. General DNA Isolation Procedures Lyse Cells • Detergent dissolves cell membrane and denatures protein. • EDTA chelates divalent cations required by nucleases. • Proteinase K degrades proteins. Organic Extraction • Uses phenol:chloroform:isoamyl alcohol • Denatures and removes proteins •Toxic Chemicals!! Nonorganic Extraction •Salt precipitation of protein • DNA precipitation • Solid Phase Extraction • Apply sample • Wash • Elute DNA • Best method! (1 X 107 cells) X (6 pg DNA/cell) X 80% yield= 48 mg DNA!!!

6. Looking at DNA: Nucleic Acid Analysis • DNA (or RNA) is characterized using several different methods for assessing quantity, quality, and molecular size. • UV spectrophotometry • Agarose gel electrophoresis • Colorimetric blotting

7. How Much DNA Do You Have? Absorbance from UV Spectrophotometry • DNA and RNA absorb maximally at a wavelength of 260 nm. • Proteins absorb at 280 nm. • Background scatter absorbs at 320 nm. • Concentration of DNA = (A260 – A320) X dilution factor X 50 µg/mL • Concentration of RNA = (A260-A320) X dilution factor X 40 µg/mL

8. Human Whole Blood DNA Lambda DNA marker Whole blood genomic DNA Lambda DNA cut with Hind III marker How Does Your DNA Look? Quality from Agarose Gel Electrophoresis

9. Pulling DNA Apart And Putting It Back Together: Denaturation and Annealing Reactions

10. DNA Likes To Find Its Perfect Match: Denaturation and Annealing of DNA

11. Hybridization Will Occur In Liquid Or On A Solid Surface

12. Melting Temperature (Tm), Salt and G + C Content

13. Basic Techniques for Analysisof Nucleic Acids • Endonuclease digestion (DNAse, RNase, restriction enzymes) • Electrophoresis (agarose and polyacrylamide gel electrophoresis) • Enzymatic modification (polymerase, kinase, phosphatase, ligase)

14. Restriction endonucleases DNA polymerases (synthesize DNA) DNA ligases (join DNA strands) Kinases (phosphorylation of 5´-ends of DNA) Phosphatases (dephosphorylate 5´-ends of DNA) Ribonucleases (digest RNA molecule. Example: RNase A) Deoxyribonucleases (digest DNA molecules) Cutting, Chewing, Tagging DNA: Nucleic Acid Modifying Enzymes

15. Restriction Endonucleases (RE) • Found only in microorganisms • Exhibit novel DNA sequence specificities • >2000 distinct restriction enzymes have been identified • Recognize symmetrical dsDNA (palindromes) • Utilized in the digestion of DNA molecules • Nomenclature: EcoRI First letter of Genus + first 2 letters of species + order of enzyme discovery E co RI

16. Restriction Enzymes Recognize Palindromes • Palindrome reads the same in both directions • BOB • “Able was I ere I saw Elba.” (Napoleon Bonapart, following his exile from the European continent to the island of Elba) • Sequences directly opposite one another on opposite strands of the ds DNA molecule

17. Enzyme produces blunt ends.. • Single strand is 5’ strand. • Single strand is 3’ strand. 4. N= any purine or pyrimidine pair. 5. Enzyme cuts 8 nts 3’ of recognition site. 6. Cuts mammalian DNA very rarely.

18. Looking At DNA: Electrophoresis • Nucleic acids are separated based on size and charge. • DNA molecules migrate in an electrical field • Employs a sieve-like matrix (THINK JELLO!) and an electrical field. • DNA is negatively charged and migrates towards the positively charged anode.

19. Gel Electrophoresis • Electrophoresis is the movement of molecules by an electric current. • Nucleic acid moves from a negative to a positive pole. • Nucleic acid has a net negative charge, they RUN TO RED

20. Principles of Gel Electrophoresis • The gel itself is composed of either agarose or polyacrylamide • Agarose is a polysaccharide extracted from seaweed • Polyacrylamide is a cross-linked polymer of acrylamide. • Acrylamide is a potent neurotoxin and should be handled with care!

21. “Submarine” Agarose Gel Electrophoresis

22. Agarose Gel Apparatus

23. Comparison Of Various Agarose Concentrations

24. 1 2 3 4 Electrophoresis Of Lambda DNA Digested Using Three Different RE • Lane 1 contains uncut lambda DNA. • Lane 2 contains lambda DNA digested by PstI. • Lane 3 contains lambda DNA digested by EcoRI. • Lane 4 contains lambda DNA digested by HindIII.

25. Restriction Enzyme Mapping • Digest DNA with a restriction enzyme. • Resolve the fragments by gel electrophoresis. • The number of bands indicates the number of restriction sites. • The size of the bands indicates the distance between restriction sites.

26. XhoI BamH1 XhoI BamH1 4.3 kb 3.7 kb 2.3 kb 1.9 kb 1.4 kb 1.3 kb 0.7 kb 4.0 kb BamH1 2.8 kb 1.7 kb 2.8 kb 1.1 kb 1.2 kb 1.2 kb 1.7 kb 1.1 kb XhoI 1.2 kb XhoI Restriction Enzyme Mapping: Circular DNA Molecules

27. Mapping DNA: Southern Blots • DNA immobilized on solid support • Detect specific DNA fragments with a DNA probe using hybridization • Ok, what the #&*^!!! is a probe? • It’s a usually a clone or amplified DNA—we’ll get there in a minute.

28. Southern Blot Hybridization: Overview

29. Southern Blot Analysis of EHV-3 DNA

30. BamHI BclI BglII EcoRI Hind III Restriction Enzyme Map of EHV-3 DNA

31. CLONE: THE NOUN AND THE VERB • To clone • produce multiple identical copies of something • A clone • identical copy, derived from single progenitor • may be DNA molecules, cells, or an organism

32. Molecular Cloning • Genetic engineering includes techniques that allow for the construction of novel DNA molecules by joining DNA sequences from different sources. Recombinant DNA Vector Clone

33. Cloning Plasmid Replicator (ori) Selectable marker Cloning site

34. Cloning into Plasmid Vectors • Cut plasmid, target DNA with RE • Treat plasmid DNA with alkaline phosphatase • Mix plasmid and target DNAs to allow annealing • Add DNA ligase

35. Transfection And Transformation: Putting Genes Back Into Cells • Calcium phosphate/chloride precipitation • Aggregates of DNA precipitate and are endocytosed • DEAE dextran • Anion binding gel that aggregates DNA • Biolistics • DNA coated onto gold microprojectiles • Electroporation • High voltage shock that makes transient DNA permeable holes in cell membranes

36. Transform bacterial cells • Treat with CaCl2 • Add media with antibiotic, incubate • Streak on selective media plate • High copy number plasmids give the best yield

37. Efficiency Of Transformation • 10% of cells treated take up DNA • 1% of cells become stable transformants • Most transfections are transient and must be forced to maintain the foreign DNA by selection pressure • Antibiotic selection • Color selection •  galactosidase gene (Lac Z) • Green fluorescent protein (GFP)

38. Selection Of Clones Containing Inserts Intact lac Z gene beta gal= blue colonies Interrupted lac Z gene No beta gal= white colonies

39. Bacteriophage Vectors • Most are derived from lambda phage • Charon 16A vectors were named after the ferryman of Greek mythology who conveyed the spirits of the dead across the River Styx

40. Yeast Vectors • Accommodate large inserts • Extremely stable • Integrate into yeast chromosome • Mini-chromosomes • Artificial chromosome • Eukaryotic system • Post-translational modifications similar to mammalian systems

41. Remember Eukaryotic Genes Contain Introns: Reverse Transcription of RNA to cDNA What about primers? Remember poly A tail? Primers are long string of Ts!

42. Provide large quantities of DNA for analysis Mapping, sequencing studies Identification, disease diagnosis Provide source of specific gene product for commercial use Production of medically important molecules Provide source of specific genes for creation of transgenic animals IS THERE A BETTER WAY TO DO SOME OF THIS? PCR and Cary Mullis What Are Cloned Genes Good For Anyway?

43. Cary Mullis and the Nobel Prize: The Basics • Knew that you could expose template DNA by boiling ds DNA to produce ss DNA • Knew that you could use primers to initiate DNA synthesis • Knew that a cheap, commercial enzyme was available (Klenow fragment of E. coli DNA polymerase)

44. Cary Mullis and PCR • Wanted a way to generate large amounts of DNA from a single copy • Initially used the “3 graduate student” method • Denaturing (unwinding) DNA • Annealing (hybridizing) primers • Extending (copying) DNA

45. THREE STEPS OF PCR • Denaturation of target (template) • Usually 95oC • Annealing of primers • Temperature of annealing is dependent on the G+C content • May be high (no mismatch allowed) or low (allows some mismatch) “stringency” • Extension (synthesis) of new strand

48. Automation of PCR • PCR requires repeated temperature changes. • The thermal cycler changes temperatures in a block or chamber holding the samples. • Thermostable (heat stable) polymerases are used to withstand the repeated high denaturation temperatures.

49. Thermostable DNA Polymerase: Yellowstone National Park And Deep Sea Vents

50. Thermostable Polymerases Taq: Thermusaquaticus (most commonly used)