1 / 21

EXU Workshop Modern Methods in Molecular Biology

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

ham
Télécharger la présentation

EXU Workshop Modern Methods in Molecular Biology

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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)

More Related