Plasmids can be constructed from natural forms, preserving only those features which help in cloning. Plasmids are constructed by excising the parts of natural plasmid by using restriction enzymes and rejoining them in correct orientations. The resultant plasmid demonstrates many desirable features for gene cloning.
Desirable features:
· Must have an origin of replication.
· Have unique recognition sites for about 20 restriction endonucleases. This means, any one restriction enzyme can cut the plasmid at only one place and only one DNA fragment can be attached for cloning.
· Have at least two genes for antibiotic resistance. This property is of utmost importance in the selection of recombinant clones.
· Have small size as far as possible because size of plasmid is very important property for their selection as cloning vectors.
Eg, pBR322
• Unique Hind III, Sal I, BamH I, and Pst I recognition sites are present in the genes for tetracycline resistance (Tetr) and ampicillin resistance (Ampr).
• The unique EcoRI site is just outside the tetracycline resistance gene.
• The origin of replication functions in the bacterium E. coli.
• The complete DNA sequence of pBR322 consists of 4,361 bp.
F COSMIDS:
Cosmids are hybrid vectors derived from the plasmids which contain cos site of the phage. The length of cosmid is about only 5Kb. Thus the length of foreign DNA which can be inserted in Cosmids is around 45Kb. The cosmid, along with inserted DNA fragment can be packed in vitro into head and tail is attached. When such a virus infects E. coli, the rDNA cyclizes through cos sites and replicates as a plasmid in the bacterial cell.
Desirable features:
· Lack the genes encoding viral proteins. Hence, the viral particles are not formed in bacteria and there will not be any cell lysis.
· Origin of replication is present.
· Marker gene for antibiotic resistance is present.
· For insertion of foreign DNA fragment, a special cleavages site is present.
· The size of cosmid is very small.
· The cosmid has cos site to phage DNA (12 base length). This helps in circularization and ligation. CLONEBANK (cDNA Library)
