1. PRACTICAL GENETIC ENGINEERING AND GENOME ANALYSIS 2015 Introductory remarks School of Biological Sciences Graduate Training Programme

2. Purpose of the course To give new Ph.D. students the knowledge required to make bold use of recombinant DNA techniques and so enable them to optimise approaches to their projects

4. Recombinant DNA technology was born out of need Jim Watson describes the world between the uncovering of the Genetic Code and the deployment of restriction enzymes http://www.dnai.org/b/index.html Manipulation  Revolution  Players  Watson  both tracks

5. The power of combining restriction enzymes & plasmids Paul Berg describes the first recombinant DNA molecule and its implications http://www.dnai.org/b/index.html Manipulation  Revolution  Players  Berg  1, 2 & 3

6. Since its birth 30 years ago, recombinant DNA technology has revolutionised biology Enabled study of viral genes in isolation from the rest of their genomes Genomic libraries used to map & sequence complex genomes Polymerase Chain Reaction allows easy isolation of sequences provided primer sequences are known This condition is met for all sequenced genomes

7. Since its birth 30 years ago, recombinant DNA technology has revolutionised biology Reverse transcriptase allows unstable RNA to be copied into stable form and studied cDNA libraries are used to isolate individual genes for functional studies Advanced expression analysis (microarray / Solexa) allows complex systems to be catalogued and system-wide perturbation studies performed

9. Lecture 1 DNA modifying enzymes for gene cloning and DNA analysis Vectors for molecular cloning Simple techniques for joining DNA molecules

10. Ability to manipulate DNA depends on access to enzymes that cleave, modify and join molecules in a specific way and having means of propagating resulting DNA : DNA modifying enzymes – understanding activities and uses Vectors – Physical and genetic properties Strategies and constraints Enzymes 1 The molecular biology toolbox

11. Enzymes 2 DNA modifying enzymes

12. SmithArberNathans Enzymes 3 The discovery of "restriction enzymes and their application to problems of molecular genetics".

13. Restriction Enzymes Type I and III – multi-subunit methylation and cleavage activities in same protein. Recognise a specific site but cleave outside at a distance, (variable in the case of Type I enzymes). 3 classes Enzymes 4 Therefore not currently exploited

14. Type II restriction endonucleases Recognise short sequences Majority are 4, 5 or 6 nucleotides in length Cleave at a specific position within or a short distance from the recognition site to produce discrete fragments. Enzymes 6

15. Need magnesium for activity Type II restriction endonucleases Methylase activity is separate Most cleave within recognition sequence Type IIs are the exception Enzymes 7

16. BamHI Enzymes 8 Palindromic?

17. Enzymes 9

18. Different enzymes produce different ends Enzymes 10

19. Different enzymes produce different ends Enzymes 11

20. Different enzymes produce different ends Enzymes 12

21. Activity can be affected by methylation Dam (GAmTC) Dcm (CCmAGG or CCmTGG) Overlapping sites (e.g. ClaI ATCGAT) CpG islands DpnI only digest methylated DNA Enzymes 13 MboI - GATC G

22. Some practical issues: Star activity- Can get off target cutting in sub-optimal conditions - HF enzymes - CutSmart buffer Temperature- not all enzymes work at 37ºC Enzymes 14

23. Double digests - Use the optimum buffer - Not always possible Definition of units – There is no universal definition of a unit Some practical issues: Enzymes 15

24. Always check your catalog before starting Enzymes 16

25. Ligases Martin Gellert Enzymes 17

26. Join duplexes via cohesive ends Joins blunt ends Seals nicks DNA Ligase Enzymes 18

27. Enzymes 19

28. DNA polymerase Enzymes 20 Require a primer Template dependent Work 5' to 3' (some have exonuclease activity) T4 PNK Reverse transcriptase Terminal transferase

29. Bacterial Tools ~5Mb of DNA ~4000 genes Can support replication and transfer of plasmids - antibiotic resistance Bacteria 1 Why do we use E.coli?

30. E.coli K12 modified to improve strain for recombinant methods hsdR system modified Bacteria 2

31. E.coli K12 modified to improve strain for recombinant methods mcrA/mcrB/mrr system modified Bacteria 3 Mcr systems digest methylated DNA

32. E.coli K12 modified to improve strain for recombinant methods recA system modified endA system modified

33. E.coli K12 modified to improve strain for recombinant methods The standard bacterial host for most recombinant DNA cloning experiments is an E. coli K12 strain with mutations in 1) endogenous restriction modification system (hsdR-) 2) homologous DNA recombination function (recA-) 3) endonuclease I activity (endA-) Bacteria 4

34. Cloning vectors are: Plasmid derived Phage derived Combination of both Cloning Vectors Vectors 1

35. Cloning vectors share four common properties : 2. Contain a genetic marker (usually dominant) for selection. 3. Unique restriction sites to facilitate cloning of insert DNA. 4. Minimum amount of nonessential DNA to optimize cloning. 1. Ability to promote autonomous replication. Vectors 2

36. Range of Cloning Vectors Plasmids M13 phage Cosmids BAC (Bacterial Artificial Chromosomes) PAC YAC (Yeast Artificial Chromosomes ) Vectors 3

37. Plasmids Plasmids ~1- 10 kb Easy to manipulate Vectors 4

38. Plasmid Biology 3 types of naturally occurring bacterial plasmids have been exploited: Virulence plasmids encoding bacterial toxins such as colicins (ColE1 plasmid ori is in cloning vectors) Conjugation plasmids (F plasmid is used to carry lacI gene and binding protein for M13 phage) Drug resistance plasmids (R plasmids are source of antibiotic resistance genes). Vectors 5

39. Modern typical multiple cloning site vector: Vectors 6

40. Vectors 7

41. Vectors 8

42. Vectors 9

43. BACs and PACs BAC=mini-F plasmid –low copy, good stability –150 - 300 kb size –low yield –size easy to manipulate –size sufficient for human DNA repeat structure Vectors 10

44. YACs 1-3 Mb possible Problems: Specific instabilities Low DNA yield Difficult to manipulate Vectors 11

45. Transformation of E. coli K-12 Two general methods for transforming bacteria. 1. A chemical method utilizing CaCl 2 and heat shock to promote DNA entry into cells. Efficiency – 10 6 -10 7 cfu/  g 2. Electroporation based on a short pulse of electric charge to facilitate DNA uptake. Efficiency – 10 8 -10 10 cfu/  g

46. Chemical method

47. Electroporation

48. Joining DNA Molecules Ends produced by different enzymes can be compatible, e.g. BamHI (G/GATCC) and BglII (A/GATCC) Options for linking DNA molecules derived with different restriction enzymes : Fill in or resect ends with DNA polymerase and perform blunt end ligation (blunt ligation is inefficient) Add linkers containing desired site and then cut with restriction enzyme to create cohesive end Add adaptors with appropriate preformed cohesive end (no restriction site cutting then necessary) Partial fill to produce cohesive ends

49. Blunt-end ligation strategy:

50. Linker ligation strategy:

51. Adaptor ligation strategy: Produces novel cohesive ends

52. Partial fill strategy BamHI XhoI G CCTAG TCGAG C Klenow + dGTP + dATP Klenow + dTTP + dCTP GGA CCTAG TCGAG CTC Ligate cut CCTAGG GGATCC CTCGAG GAGCTC

53. GGA CCTAG TCGAG CTG Product is not cleavable by either BamHI or XhoI

54. Complete fill strategy HindIII Vector Digest A TTCGA AGCTT A TTCGA AAGCT AGCTT TCGAA Fill AAGCT TTCGA AGCTT TCGAA NheI Ligate

55. PCR Techniques > Amplifying > Making copies PCR animation Interviews

57. Use PCR to amplify desired DNA with primers that contain appropriate restriction sites BamHI EcoRI BamHI EcoRI BamHI EcoRI

58. Use Topoisomerase cloning method – takes advantage of extra A residue added by Taq polymerase and uses vector with activated ends

59. Topo TA Topo blunt

60. Recombinant DNA construction independent of restriction sites PCR technology provides one option for creating in vitro new molecules independent of restriction site position – overlap extension technique. 5’ PCR 5’ PCR Combine PCR products

61. 5’ Denature and anneal Extend 3’ ends 5’ PCR 5’ Final recombinant DNA product

62. Usual PCR related problems to consider – mutation during synthesis, mispriming Appropriate up to several kilobases but not easy for very large fragments

63. Now to the lab……………….. nick.mullin@.ed.ac.uk