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Chapter 4

Chapter 4. Molecular Cloning Methods. Gene Cloning. The Role of Restriction Endonuclease. Vectors. Plasmids : carriers that allow replication of recombinant DNAs. Action of DNA ligase. r: resistant s: snsitive. lacZ ’: b -galactosidase

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Chapter 4

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  1. Chapter 4 Molecular Cloning Methods

  2. Gene Cloning The Role of Restriction Endonuclease

  3. Vectors Plasmids : carriers that allow replication of recombinant DNAs

  4. Action of DNA ligase r: resistant s: snsitive

  5. lacZ’: b-galactosidase X-gal: synthetic substrate for b-galactosidase MCS: multiple cloning sites Blue/white color selection

  6. Enhance of the cloning efficiency: dephosphorylation of the 5’-phophate in the vector by alkaline phosphatase to prevent self-ligation

  7. Phages as Vectors • replacement vectors *up to 20 kb of DNA can be cloned. *construction of genomic libraries *Charon 4: (12 kb< inserts <20 kb) Cosmids * cohesive ends of l phage DNA * plasmid origin of replication * up to 40-50 kb of DNA can be cloned

  8. M13 phage vectors * derived from filamentous phage M13 * contains lacZ’gene and MCS * DNA can be cloned in a single-strand form (introduction of site-specific mutation)

  9. Phagemids * contains ori of filamentous phage f1, lacZ’gene and MCS * DNA can be cloned in a single-strand form via the help with f1 helper phage * contains T7 and T3 phage promoter for phage RNA polymerase (in vitro transcription to produce RNA transcripts)

  10. Identifying a specific clone with a specific probe • Poly(oligo)nucleotide probes • Antibodies • To probe the gene what we want: • Homologous gene from another organism • *Low/high stringency for hybridization of oligonucleotide probes to targets • *High stringency: high temperature, high organic concentration, • low salt concentration • b. Degenerate primers from known amino acid sequence of target protein U G U UGG AUG UUC AAA AAC GA Trp Met Phe Lys Asn Glu

  11. The Polymerase Chain Reaction (PCR) *Kary Mullis in the 1980s *Taq polymerase from Thermus aquaticus *Thermal cycler 95oC 40oC 72oC 20x

  12. cDNA Cloning (complementary or copy DNA) Nick translation using E.Coli DNA polymerase I with 5’ 3’ exonuclease activity

  13. Nick translation

  14. BamH1 Using RT-PCR in cDNA Cloning HindIII

  15. 5’-RACE of a cDNA Rapid Amplification of cDNA Ends (completing of the 5’end of the mRNA)

  16. Methods of Expressing Cloned Genes pUC and pBS vectors—lac promoter (in frame)

  17. ptrpL 1 vector—trp operator/promoter (strong promoter) SD: Shine-Dargarno ribosome binding site

  18. Expression of Hisx6-tagged fusion protein using pTrcHis vector EK: enterokinase (a protease) cutting site

  19. Expression of fusion protein using lgt11 vector in E. Coli

  20. Eukaryotic Expression Systems Expression in E.Coli: Inclusion bodies No posttranslational modification Expression in yeast Expression in caterpillar using Baculovirus-derived vectors (10% in dry mass, polyhedrin)

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