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

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lecture 4
Lecture 4
  • Recombinant DNA technology
  • Gene expression in prokaryotic systems
slide2

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
slide3

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
slide4

Restriction enzymes:

Host restriction (and modification)

slide7

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

slide11

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
slide13

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

slide24

Characterization of inserts: Nucleic acids hybridization

Edwin Southern 1975, “Southern blot”

slide26

Variations on Southern blotting

Combine with restriction enzyme digestion and gel electrophoresis

Northern blots

Western blots

Southwestern blots

slide28

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

slide29

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

slide35

Conjugation

Lederberg Monod

  • F- to F+
  • 100 minutes
  • 4000 genes
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