EXU Workshop Modern Methods in Molecular Biology

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EXU WorkshopModern Methods in Molecular Biology

• 12.5.-16.5.2008

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Kary Mullis

• The PCR technique was patented by Cetus
  Corporation, where Mullis worked when he invented
  the technique in 1983. There have been several
  high-profile lawsuits related to the technique.
  The pharmaceutical company Hoffmann-La Roche
  purchased the rights to the patents in 1992 and
  currently holds those that are still protected.
• He was awarded the Nobel Prize in Chemistry in
  1993 for his invention, seven years after he and
  his colleagues at Cetus first put his proposal to
  practice.
• The Taq polymerase enzyme was also covered by
  patents. A related patent battle over the Taq
  polymerase enzyme is still ongoing in several
  jurisdictions around the world between Roche and
  Promega.

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Herbert Boyer, Stanley Cohen
conference in Hawaii in 1972 Bacterial
plasmids-circular segments of DNA Boyer was a
biochemist and genetic engineer at UCSF Cohen was
an associate professor of medicine at Stanford
University
1973 - birth of gene engineering invented the
technique of DNA cloning, which allowed genes to
be transplanted between different biological
species.
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The birth of gene engineering.

• Boyer's lab isolated an enzyme that could be
  used to cut strings of DNA into precise and
  "cohesive" segments
• Cohen developed a method to introduce
  antibiotic-carrying plasmids
• into certain bacteria
• method of isolating and cloning genes carried by
  the plasmids
• Boyer and Cohen decided to pool their resources
  Boyer's enzyme would allow Cohen to introduce
  specific DNA segments to plasmids, and use those
  plasmids as a vehicle for cloning precise,
  previously targeted strands of DNA
• Within four months, the joint effort of Boyer's
  and Cohen's labs had succeeded in cloning
  predetermined patterns of DNA.

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Plasmid purification

• Birnboim HC, Dolly J. (1979). A rapid alkaline
  extraction procedure for screening recombinant
  plasmid DNA. Nucl. Acids Res. 7(6) 15131523.
• Holmes D S Quigley M. (1981). A rapid boiling
  method for the preparation of bacterial plasmids.
  Anal. Biochem. 114193-197

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Alkaline lysis

• Lysing (RNase)
• Alkaline denaturing
• Neutralizing
• Remove proteins, gDNA
• (Phenol/chloroform extraction, alcohol
  precipitation.)
• Column purification

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Boiling lysis

• Lysing (RNase)
• Boiling
• Remove proteins, gDNA
• (Phenol/chloroform extraction, alcohol
  precipitation.)
• Column purification

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BL21 (DE3)pLysS F-, ompT, hsdSB(rB- mB-), gal,
dcm, (DE3), pLysS(CamR)

• Proteases Lon
• OmpT
• heterologous proteins
• recipient cells for unmodified or foreign DNA
• hsd genes - DNA restriction-modification (R-M)
  systems
• are usually restrictionless mutants
• mutants may either modify and have the phenotype
  (r- m)
• or fail to modify and have the phenotype (r- m-)
• R-M system consists of 3 genes
• hsdS for specificity, hsdR for restriction, hsdM
  for modification
• ?DE3 lysogen
• lacUV5-T7 RNA pol IPTG inducible

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pLysS

• T7 lysosyme
• reduce basal expression of heterologous proteins
  (toxic)
• p15A plasmidcompatible with ColE1, pMB

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SDS-PAGE

• U. K. Laemmli (1970). "Cleavage of structural
  proteins during the assembly of the head of
  bacteriophage T4". Nature 227 (5259) 680-685.
• separate proteins according to their
  electrophoretic mobility (function of length of
  polypeptide chain or molecular weight)

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Composition

• Stacking gel large pore polyacrylamide gel (4)
• Tris buffer pH 6.8
• Resolving gel small pore polyacrylamide gel
  (12)
• Tris buffer pH 8.8
• proteins separate according to their size (14-66
  kD)
• Electrophoresis and staining
• denatured proteins migrate depending on their
  size (molecular weight)
• stained (Coomassie Brilliant Blue)
• 40 kDa

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PIManT (E.C.2.4.1.57)

• JBC 277(35) 31335-31344 (2002)
• JBC 282(28) 20705-20714 (2007)