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

Lecture 4

  • Recombinant DNA technology

  • Gene expression in prokaryotic systems


Lecture 4

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


Lecture 4

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


Lecture 4

Restriction enzymes:

Host restriction (and modification)


Lecture 4

At the molecular level


Lecture 4

Host modification


Lecture 4

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


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Proteins recognize palindrome to bind tightly to DNA


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Restriction enzymes and restriction sites: use 1


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Use 2: Molecular cloning


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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


Lecture 4

Cloning into plasmids and host


Lecture 4

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


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Construction of ‘ideal’ vectors: Plasmid


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Construction of ‘ideal’ vectors: pUC series


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Need for bigger inserts: Lambda phage vector


Lecture 4

Larger inserts from euk genes: cDNA by reverse transcription


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Characterization of inserts: Nucleic acids hybridization


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Hybridization to inserts within lambda genome


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Characterization of inserts: Nucleic acids hybridization

Edwin Southern 1975, “Southern blot”


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Southern blot (cont.)


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Variations on Southern blotting

Combine with restriction enzyme digestion and gel electrophoresis

Northern blots

Western blots

Southwestern blots


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Gel electrophoresis, blot and probe


Lecture 4

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


Lecture 4

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


Lecture 4

Second generation nucleic acids probes through oligonucleotide synthesis


Lecture 4

Manipulating eukaryotes, bridge from prokaryotes


Lecture 4

Conjugation

Lederberg Monod

  • F- to F+

  • 100 minutes

  • 4000 genes


Lecture 4

Tools for genetics: conditional mutations


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