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EXU Workshop Modern Methods in Molecular Biology

EXU Workshop Modern Methods in Molecular Biology. 12.5.-16.5.2008. Kary Mullis.

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EXU Workshop Modern Methods in Molecular Biology

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  1. EXU WorkshopModern Methods in Molecular Biology 12.5.-16.5.2008

  2. 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.

  3. 35 cycles: 95 °C / 1’ 64 °C / 1’ 72 °C / 1.5’

  4. pimA1 gene 236 = 6.87×1010 copies

  5. 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.

  6. 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.

  7. Plasmid purification • Birnboim HC, Dolly J. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl. Acids Res.7(6): 1513–1523. • Holmes D S & Quigley M. (1981).A rapid boiling method for the preparation of bacterial plasmids. Anal. Biochem. 114:193-197

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

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

  10. BL21 (DE3)pLysS: F-, ompT, hsdSB(rB- mB-), gal, dcm, (DE3), pLysS(CamR) • Proteases: Lon OmpT • heterologous proteins • recipient cells for unmodified or foreignDNA • 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 thephenotype (r- m-) • R-M system consists of 3 genes: • hsdS for specificity, hsdR for restriction,hsdM for modification • λDE3 lysogen: • lacUV5-T7 RNA pol; IPTG inducible

  11. T7 lysosyme reduce basal expression of heterologous proteins (toxic) p15A plasmid:compatible with ColE1, pMB pLysS

  12. SDS-PAGE • U. K. Laemmli (1970). "Cleavage of structural proteins during the assembly of the head of bacteriophage T4". Nature227 (5259): 680-685. • separate proteins according to their electrophoretic mobility (function of length of polypeptide chain or molecular weight)

  13. 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

  14. PIManT (E.C.2.4.1.57) • JBC 277(35): 31335-31344 (2002) • JBC 282(28): 20705-20714 (2007)

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