Gene Cloning

1. Gene Cloning

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

5. 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.

6. Sources of DNA for Cloning 1) Chromosomal DNA 2) Extrachromosomal DNA 3) RNA converted to cDNA 4) PCR-amplified DNA

7. RNA converted to cDNA

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 EcoRI E = genus (Escherichia) co = species (coli) R = strain I = # of enzyme

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

13. Cloning vectors

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)

15. A plasmid vector for cloning

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

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)

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

24. BAC vector oriS and oriE mediate replication parA and parB maintain single copy number ChloramphenicolR marker

25. YAC vector Capable of carrying inserts of 200 - 2000 kbp in yeast

26. What determines the choice vector? insert size

27. Expression vector

29. Host organism bacterial host � E. coli eukaryotic host � yeast (Saccharomyces cerevisiae) other hosts � other yeasts, insect cells, etc.

30. Method of introducing DNA into the host cell

31. Method of introducing DNA into the host cell

32. Method of introducing DNA into the host cell

35. 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

40. 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

41. Blue/White Color Screening

42. Selecting Colonies with Recombinant Plasmids

43. Colony hybridization DNA probe available? part of same gene orthologue from another species synthetic oligonucleotide

44. End