Lecture 4

1. Lecture 4 • Recombinant DNA technology • Gene expression in prokaryotic systems

2. Molecular biology: Recombinant DNA technology. Key technique • Summer ‘71 SV40 DNA to E. coli experiment postponed • Feb ‘75 Asilomar Conference: Most rDNA work should continue, with safeguards

3. Molecular biology: Recombinant DNA technology • Use naturally occurring proteins and nucleic acids, • Molecular biology, as • Molecular tools • Manipulate for desired effect and product • Reproducible, archivable

4. Restriction enzymes: Host restriction (and modification)

5. At the molecular level

6. Host modification

7. and Restriction enzymes and recognition sites Palindromes: 55, 212, 1331, 45654 pop, level, racecar Madam, I’m Adam Was it a rat I saw? A man, a plan, a canal, panama GGATCC CCTAGG

8. Proteins recognize palindrome to bind tightly to DNA

9. Restriction enzymes and restriction sites: use 1

10. Use 2: Molecular cloning

11. Basic science discovery of recombinant DNA technology • Nov 1972 Honolulu Meeting on plasmids • Boyer- bacterial enzymes which cut DNA at specific sites • Cohen- collaboration • 1973- series of expts resulting in method to select and replicate specific foreign genes in bacteria • Feb 1975 Asilomar in Pacific Grove, CA; goal to estimate risk of biohazard and formulate guidelines • Dec 1980 First of three patents on gene cloning to Stanford and UC • April 1976 Genentech incorporated (Boyer) • 1977 Rutter et al cloned rat insulin gene • 1981 Founded Chiron • 1986 First recomb vaccine to receive FDA approval; • Chiron-Merck hepB vaccine • http://bancroft.berkeley.edu/Exhibits/Biotech/25

12. Cloning into plasmids and host

13. Vector and insert size M13 1.5kb Bias of sequences Plasmids 3.5-20kb Size instability Lambda phage 10-15kb Size limitation Cosmids 45kb Size limitation BAC 100-300kb YAC 1Mb Up to 60% chimera

14. Construction of ‘ideal’ vectors: Plasmid

15. Construction of ‘ideal’ vectors: pUC series

16. Need for bigger inserts: Lambda phage vector

18. Larger inserts from euk genes: cDNA by reverse transcription

22. Characterization of inserts: Nucleic acids hybridization

23. Hybridization to inserts within lambda genome

24. Characterization of inserts: Nucleic acids hybridization Edwin Southern 1975, “Southern blot”

25. Southern blot (cont.)

26. Variations on Southern blotting Combine with restriction enzyme digestion and gel electrophoresis Northern blots Western blots Southwestern blots

27. Gel electrophoresis, blot and probe

28. Hybridization results: Comparison of signals Southern: single copy INO2 gene. 5ug yeast genomic DNA with biotinylated cRNA probe. H3, R1, Sal1 with one minute exposure Northern: TCM1 expression. 5, 2.5, 0.625, 0.313 and 0.156ug yeast genomic DNA with A/B DIG-label; C/D biotinylated; E 32P Exposure times: A 5’; B 25’; C 5’; D 25’; E 72hrs

29. Zoo blots: Comparative genomics, pre-genomes 250 bp CSB probe Low stringency: 2xSSC/50C/15min Higher stringency: 1xSSC/50C (-chicken and xenopus) Higher stringency: only primates left

30. Second generation nucleic acids probes through oligonucleotide synthesis

31. Manipulating eukaryotes, bridge from prokaryotes

35. Conjugation Lederberg Monod • F- to F+ • 100 minutes • 4000 genes

37. Tools for genetics: conditional mutations