Lecture 4
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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

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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


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


Restriction enzymes:

Host restriction (and modification)


At the molecular level


Host modification


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


Proteins recognize palindrome to bind tightly to DNA


Restriction enzymes and restriction sites: use 1


Use 2: Molecular cloning


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


Cloning into plasmids and host


Vector and insert size

M13 1.5kb Bias of sequences

Plasmids 3.5-20kb Size instability

Lambda phage 10-15kbSize limitation

Cosmids 45kbSize limitation

BAC 100-300kb

YAC 1Mb Up to 60% chimera


Construction of ‘ideal’ vectors: Plasmid


Construction of ‘ideal’ vectors: pUC series


Need for bigger inserts: Lambda phage vector


Larger inserts from euk genes: cDNA by reverse transcription


Characterization of inserts: Nucleic acids hybridization


Hybridization to inserts within lambda genome


Characterization of inserts: Nucleic acids hybridization

Edwin Southern 1975, “Southern blot”


Southern blot (cont.)


Variations on Southern blotting

Combine with restriction enzyme digestion and gel electrophoresis

Northern blots

Western blots

Southwestern blots


Gel electrophoresis, blot and probe


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


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


Second generation nucleic acids probes through oligonucleotide synthesis


Manipulating eukaryotes, bridge from prokaryotes


Conjugation

Lederberg Monod

  • F- to F+

  • 100 minutes

  • 4000 genes


Tools for genetics: conditional mutations


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