Cloning and Mutagenesis

HAMID M. SADEGHI

Pharmaceutical Research Center, Faculty of Pharmacy, Isfahan University of Medical Sciences, Isfahan, I.R. Iran

Mutagenesis

Some Necessary Tools of Genetic Engineeirng
•Restriction enzymes
•Plasmids

Restriction endonucleases cleave only double stranded DNA by recognition of specific sequences within the DNA molecule.

•Protruding Ends (also referred to as "sticky" ends or "staggered" ends).
A. Protruding 5' (PO4) ends: occurs when the enzyme cuts to the left of the central axis of symmetry
5'......G*AATTC......3'
3'......CTTAA*G......5'
5'......GOH PO4AATTC......3'
3'......CTTAAPO4 OHG......5'

Ligase

The reaction requires the expenditure of two high energy phosphate bonds. Notice that the phosphate on the 5' end of the nick is used to make the phosphodiester bond. ATP provides only energy, not a phosphate.

Plasmids

Plasmids are molecules of DNA that are found in bacteria separate from the bacterial chromosome. They:
•are small (a few thousand base pairs)
•usually carry only one or a few genes
•are circular
•have a single origin of replication
Plasmids are replicated by the same machinery that replicates the bacterial chromosome. Some plasmids are copied at about the same rate as the chromosome, so a single cell is apt to have only a single copy of the plasmid. Other plasmids are copied at a high rate and a single cell may have 50 or more of them.

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