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2. Gene Cloning 3. Cloning - a definition

  • From the Greek - klon, a twig
  • An aggregate of the asexually produced progeny of an individual;a group of replicas of all or part of a macromolecule (such as DNA or an antibody)
  • An individual grown from a single somatic cell of its parent & genetically identical to it
  • Clone: a collection of molecules or cells, all identical to an original molecule or cell

4. DNA CLONING A method for identifying and purifying a particular DNA fragment (clone) of interest from a complex mixture of DNA fragments, and then producing large numbers of the fragment (clone) of interest. 5. Gene cloning

  • When DNA is extracted from an organism, all its genes are obtained
  • In gene (DNA) cloning a particular gene is copied (cloned)

6. Why Clone DNA?

  • A particular gene can be isolated and its nucleotide sequence determined
  • Control sequences of DNA can be identified & analyzed
  • Protein/enzyme/RNA function can be investigated
  • Mutations can be identified, e.g. gene defects related to specific diseases
  • Organisms can be engineered for specific purposes, e.g. insulin production, insect resistance, etc.

7. Sources of DNA for Cloning

  • 1)Chromosomal DNA
  • 2)RNA converted to cDNA
  • 3)PCR-amplified DNA

8. PCR-amplified DNA 9. Cloning Tools

  • Restriction endonucleases
  • Ligase
  • Vectors
  • Host
  • Methods for introducing DNA into a host cell

10. Cutting DNA

  • Restriction endonucleases (restriction enzymes)
    • sticky ends
    • blunt ends
  • Nomenclature
    • Eco RI
    • E= genus ( Escherichia )
    • co= species ( coli )
    • R = strain
    • I = # of enzyme

11. Blunt & Sticky ends 12. Pasting DNA

  • Complementary ends (sticky ends) H-bond
  • Ligase forms phosphodiester bond to seal strands together.

13. Cloning vectors allowing the exogenous DNA to be inserted, stored, and manipulated mainly at DNA level.1Plasmid vectors 2Bacteriophage vectors 3Cosmids4BACs & YACs 14. Plasmid vectors

  • Advantages:
    • Small, easy to handle
    • Straightforward selection strategies
    • Useful for cloning small DNA fragments
    • (< 10kbp)
  • Disadvantages:
    • Less useful for cloning large DNA fragments
    • (> 10kbp)

Plasmid vectors are double-stranded, circular, self-replicating, extra-chromosomal DNA molecules. 15. A plasmid vector for cloning

  • Contains an origin of replication,allowing for replication independent of hosts genome.
  • ContainsSelective marker s: Selection of cells containing a plasmid
  • twin antibiotic resistance
  • blue-white screening
  • Contains amultiple cloning site( MCS )
  • Easy to be isolatedfrom the host cell.

16. Plasmid vectors 17. Bacteriophage vectors

  • Advantages:
    • Useful for cloning large DNA fragments
    • (10 - 23 kbp)
    • Inherent size selection for large inserts
  • Disadvantages:
    • Less easy to handle

18. vectors

  • Left arm :
    • head & tail proteins
  • Right arm :
    • DNA synthesis
    • regulation
    • host lysis
  • Deleted central region :
    • integration & excision
    • regulation

19. 20. Cosmid vectors

  • Advantages:
    • Useful for cloning very large DNA fragments
    • (32 - 47 kbp)
    • Inherent size selection for large inserts
    • Handle like plasmids
  • Disadvantages:
    • Not easy to handle very large plasmids (~ 50 kbp)
    • combine the properties of plasmid vectors with theuseful properties of the lcossite

21. 22. ZAP 23. BACs and YACs

  • Advantages:
    • Useful for cloning extremely large DNA fragments
    • (100 - 2,000 kbp)
    • This is very important for genome sequencing projects
  • Disadvantages:
    • Not easy to handle extremely large DNA molecules

BACs:B acterialA rtificialC hromosome s YACs:Y eastA rtificialC hromosome s 24. BAC vector

  • ori S andori E mediate replication
  • par A andpar B maintain single copy number
  • Chloramphenicol Rmarker

25. YAC vector

  • Capable of carrying inserts of 200 - 2000 kbp in yeast

telomere telomere centromere URA3 ARS HIS3 replication origin markers large inserts 26. What determines the choice vector?

  • insert size
  • vector size
  • restriction sites
  • copy number
  • cloning efficiency
  • ability to screen for inserts
  • what down-stream experiments do you plan?

27. Expression vector 28. expression vector pSE420

  • polylinker: insert desired DNA
  • amp resistance
  • trcpromoter
  • lacO(operator)
  • Shine-Dalgarno (S/D) site
  • (ribosome binding)
  • T1, T2 transcription terminators
  • lacI( lacrepressor)

growth inducer added cloned gene expressed; product produced (Fig. 31.4, p. 1002, Madigan et al.) Btech10 29.

  • insertion of foreign DNA atBam HI site
  • tet resistance gene inactivated
  • transformants carrying foreign DNA are amp resistant but tetracycline sensitive

(Fig. 10-42, p. 309, Madigan et al.) transformation:transfer of genetic information via free DNA Btech6 How to clone DNA 30. How to clone DNA

  • Isolation of cloning vector(bacterial plasmid)& gene-source DNA (gene of interest)
  • Insertion of gene-source DNA into the cloning vector using the same restriction enzyme; bind the fragmented DNA with DNA ligase
  • Introduction of cloning vector into cells (transformation by bacterial cells)
  • Cloning of cells (and foreign genes)
  • Identification of cell clones carrying the gene of interest

31. Screening of the clone

  • The medium in this petri dish contains the antibiotic Kanamycin
  • The bacteria on the right contain Kanr, a plasmid that is resistant to Kanamycin, while the one on the left has no resistance
  • Note the difference in growth

32. Blue/White Color Screening lacZ lacZ insert functional enzyme nonfunctional enzyme X-gal product X-gal product 33. Selecting Colonies with Recombinant Plasmids 34. Colony hybridization

  • DNA probe available?
    • part of same gene
    • orthologue from another species
    • synthetic oligonucleotide

Figure 6.12 35. bacteriophage lambda as a cloning vector (Fig. 10.44, p. 311, Madigan et al.) transduction:transfer of host genes from one cell to another by a virus Btech7 36. Other methods for introducing DNA

  • electroporation:the use of an electric pulse to enable cells to take up DNA
  • millisecond-length pulses open small pores in cell membranes
  • DNA can move into/out of the cells via pores

cell plasmid transformant plasmid donor desired transformant microprojectile gun Btech8 37. transgenic plants may be produced with binary vector system inAgrobacterium tumefaciens (Fig. 31.11, p. 1014, Madigan et al.)

  • (a) generalized plant cloning vector
    • ends of T-DNA (red)
    • ori( E. coli ),ori( A. tumefaciens )
    • resistance markers (kan, spec)

(b) can clone inE. coli;transfer toA. tumefaciensby conjugation (c) D-Ti = engineered Ti (to remove pathogenesis genes) (d) D-Ti will mobilize T-DNA of vector-> plant cells grown in tissue culture (e) whole plants can be regenerated from recombinant cell Btech15 38. End ARRIVIDIERCI