DEPARTMENT OF MICROBIOLOGY, S.M. JOSHI COLLEGE, HADAPSAR, PUNE Cloning Genes BY NIKAM C.D. ASSISTANT PROFESSOR, DEPARTMENT OF MICROBIOLOGY, S.M. JOSHI COLLEGE, HADAPSAR, PUNE

Cloning Genes Gene cloning: amplifying a specific piece of DNA via a bacteria cell Cloning vector: a replicating DNA molecule attached with a foreign DNA fragment to be introduced into a cell Has features that make it easier to insert DNA and select for presence of vector in cell. Origin of replication Antibiotic resistance gene Cloning site

19.5 An idealized cloning vector has an origin of replication, one or more selectable markers, and one or more unique restriction sites.

Cloning Genes Plasmid vectors Linkers: synthetic DNA fragments containing restriction sites Transformation of host cells with plasmids Selectable markers are used to confirm whether the cells have been transformed or not.

19. 6 The pUC19 plasmid is a typical cloning vector 19.6 The pUC19 plasmid is a typical cloning vector. It contains a cluster of unique restriction sites, an origin of replication, and two selectable markers— an ampicillin-resistance gene and a lacZ gene.

19.7 A foreign DNA fragment can be inserted into a plasmid with the use of restriction enzymes.

19.8 The lacZ gene can be used to screen bacteria containing recombinant plasmids. A special plasmid carries a copy of the lacZ gene and an ampicillin- resistance gene. [Photograph: Cytographics/Visuals Unlimited.]

19.8 (part 2) The lacZ gene can be used to screen bacteria containing recombinant plasmids. A special plasmid carries a copy of the lacZ gene and an ampicillin-resistance gene. [Photograph: Cytographics/Visuals Unlimited.]

TA cloning of PCR product

Bacterial transformation Use modified strain of E. coli Restriction system knocked out Recombination system limited LPS is truncated Pores are made in cell membrane by treatment with calcium chloride. Cells must be kept cold to prevent pores from closing

Bacterial transformation DNA added to cells forms intimate interaction with membrane Briefly moved to 42° C to heat shock cells Stresses them and causes them to take up DNA

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