1. Chapter 4 Molecular Cloning Methods

2. Gene Cloning The Role of Restriction Endonuclease

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

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

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

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

8. 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  phage DNA * plasmid origin of replication * up to 40-50 kb of DNA can be cloned

10. 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)

11. 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)

12. Identifying a specific clone with a specific probe 1.Poly(oligo)nucleotide probes 2.Antibodies To probe the gene what we want: a.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

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

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

15. Nick translation

16. Using RT-PCR in cDNA Cloning BamH1 HindIII

17. 5’-RACE of a cDNA Rapid Amplification of cDNA Ends (completing of the 5’end of the mRNA)

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

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

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

21. Expression of fusion protein using gt11 vector in E. Coli

23. 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)