DNA Techniques

1. DNA Techniques • Recombinant DNA • DNA Sequencing • Gel Electrophoresis • DNA Amplification • DNA Fingerprinting • Gene Therapy by no means all...

2. Recombinant DNA • Getting genes from one organism into another • Target gene vs. vector • Restriction endonucleases • tool for cutting DNA prior to insertion

3. Restriction Endonucleases • Naturally occurring bacterial enzymes • Cut DNA (of attacking viruses?) • break phosphodiester bonds • Named for organism of origin • EcoRI came from E. coli strain RI

4. Restriction Endonucleases • “Restriction” = restricted to cutting at very specific sites • palindromic DNA sequences • inverted repeats • flush cutters and staggered cutters

5. Sample of Restriction Enzymes and Cutting Sites EcoRI 5’G A A T T C C T T A A G 5’ Staggered cut EcoRII 5’ G C C T G G C C G G A C C G 5’ Staggered cut

6. A A G C T T T T C G A A 5’ A A G C T T T T C G A A 5’ Staggered Cuts vs. Flush Cuts 5’ G T Py Pu A C C A Pu Py T G 5’ G T Py Pu A C C A Pu Py T G "sticky ends"

7. Sticky Ends • Unpaired bases have affinity for other unpaired bases • will reform hydrogen bonds w/ complementary bases

8. --C G C T A C C A A G C T T A C C G A-- --G C G A T G G T T C G A A T G G C T-- --G C T A C C A A G C T ----- A G C T T A C C G -- --C G A T G G T T C G A ----- T C G A A T G G C -- A G C T -------- -------- T C G A Recombinant DNA

9. Cloning • Making multiple copies of a target gene • Generally use bacteria as the “factory” • reproduce incredibly quickly • have natural VECTOR • Vectors carry the target gene

10. Cloning • Vectors • segments of DNA that carry target gene; allow reproduction of gene • plasmids • phages • cosmids • part viral, part plasmid • BACs - bacterial artificial chromosomes • YACs - yeast artificial chromosomes

11. Cloning chromosome Target gene Need restriction enzyme site on either side of target gene as well as w/in vector! Vector

12. Cloning • Use SAME restriction endonuclease to cut out target gene and open vector • Must use an enzyme that produces sticky ends! • Will have complementary sticky ends on target and vector

13. Cloning Bacteria will now produce target gene product Transform bacteria w/ vector

14. Cloning • Interferon was the first human protein to be cloned this way • Human proteins produced w/ recombinant DNA technology: • insulin • human growth hormone • superoxide dismutase (heart attack) • Factor VIII (hemophilia)

15. Human proteins produced w/ recombinant DNA technology: Fertility hormones Interleukin-2 (kidney disease) erythropoietin (anemia) lung surfactant protein (premature infants) DNase (cystic fibrosis) Renin inhibitor (high blood pressure) colony stimulating factor (post chemo-therapy)

16. Transgenic organisms • Genes may be transferred from eukaryotic one organism to another • plants that glow in the dark • transferred luciferase gene • plants w/ herbicide resistance • cows w/ human lactoferrin gene • binds iron, prevents infection, (infant formula) • goats w/ human tissue plasminogen activator • breaks up blood clots

17. Gel Electrophoresis • Used to separate molecules • Protein, RNA, DNA • Separation based on size of molecules, charge, shape, etc. • Necessary for Southern blots, DNA sequencing, DNA fingerprinting, RFLP analysis, many other applications

18. - +

19. Protein gels • Used to identify genotypes • Electrophoretic patterns inherited as alleles • Electromorphs or allelomorphs • A : one protein • a : slightly different protein

20. Protein gels 1 2 3 Lane 1: homozygote for slow allele Lane 2: heterozygote for both alleles Lane 3: homozygote for fast allele

21. RFLPs:Restriction Fragment Length Polymorphisms • Differences in restriction sites; DNA from different sources cut into different sizes • Flush or staggered cutters • Quantify differences between species; relatedness • Identify source of tissue samples (forensics, etc.) • Markers for genetic disease

22. RFLPs

23. RFLPs

24. RFLPs

25. RFLPs as markers Normal b-globin gene: Mst II site Sickle cell b-globin gene: no restriction site

26. RFLPs as markers SS Ss ss

27. RFLPs as markers Ind. A Ind. B SS Ss ss

28. Southern Blots • Way to visualize specific DNA segments • Transfers DNA from gel to nitrocellulose filter • “Stains” specific DNA segment w/ radioactive, single-stranded DNA probe • Northern blots: RNA • Western blots: protein

29. Southern Blot If stain for non-specific DNA, see a “smear” 1. Heat to separate strands 2. Add radioactive probe 3. Cool, probe binds to complementary areas 4. Place filter on X-ray film, develop, see bands!

30. DNA Fingerprinting • Very specific form of RFLP analysis • allows differentiation even if no differences in restriction sites • Uses VNTRs • variable number of tandem repeats • short, repeated sequences (~ 4 bases) • GGACGGACGGACGGACCC TGCCTGCC TGCCTG • non-coding, highly variable between individuals

31. DNA Fingerprinting - Parentage tests mother child Mr. A Mr. B Mr. C

32. DNA Sequencing • Sanger or Dideoxynucleotide method • Uses base analogues that are “chain terminators” • Depends on ability of electrophoresis to distinguish between DNA segments that differ in length by only one base

33. DNA Sequencing • Dideoxynucleotides • sugar missing two oxygens • oxygen on 3’ carbon missing • no new nucleotide may bind • chain terminator • Copy DNA template using ddnucleotides

34. DNA Sequencing • Run four DNA synthesis reactions • one with 4 normal bases and some ddATP • one with 4 normal bases and some ddTTP • one with 4 normal bases and some ddCTP • one with 4 normal bases and some ddGTP • Run a gel; each rxn in a lane (4 lanes)

35. 3’---------T A C T A T G C C A G A 5’ 5’ primer ddATP rxn: 5’ primer A T G A T A 5’ primer A T G A (Sometimes the entire segment is replicated. This happens in all the rxns and can be ignored.) 5’ primer A Three different segments produced: 1 base, 4 bases, and 6 bases in length ignoring primer.

36. 3’---------T A C T A T G C C A G A 5’ 5’ primer ddTTP rxn: 5’ primer A T G A T A C G G T C T 5’ primer A T G A T A C G G T 5’ primer A T G A T 5’ primer A T

37. 3’---------T A C T A T G C C A G A 5’ ddATP A T G A T A A T G A A ddTTP A T G A T A C G G T C T A T G A T A C G G T A T G A T A T ddGTP A T G A T A C G G A T G A T A C G A T G ddCTP A T G A T A C G G T C A T G A T A C

38. 3’---------T A C T A T G C C A G A 5’ “Read” sequence: Run on a gel: T C T G G C A T A G T A 5’ A T G A T A C G G T C T A T G A T A C G G T C A T G A T A C G G T A T G A T A C G G A T G A T A C G A T G A T A C A T G A T A A T G A T A T G A A T G A T A

39. Polymerase Chain Reaction (PCR) • Used to amplify DNA samples • Small tissue sample --> enough DNA to do fingerprinting, etc. • Repeated cycles of DNA replication • Uses Taq polymerase

40. PCR heat Cool in presence of primer (DNA oligonucleotide) nucleotides, polymerase Repeat cycle many times