Origin of replication (Ori)
• This is a specific sequence from where replication starts, when any piece of foreign DNA is linked to this sequence.
• The replication occurs inside the host cells. This sequence is also responsible for controlling the copy number of linked DNA.
• A prokayotic DNA has single origin of replication, while eukaryotic DNA may have more than one origin of replication.
• The vector also requires a selectable marker (antibiotic resistance gene) to identify and eliminate non transformants and selectively permit the growth of the transformants.[userpro_private]
• Transformation is a process through which a piece of DNA is introduced in a host bacterium.
• Generally, the genes encoding resistance to antibiotics such as tetracycline, ampicillin, kanamycin or chloramphenicol etc. are useful selectable markers for E. coli.
• A molecular marker is a fragment of the DNA molecule that is associated with certain trait(s) in an organism.
• Markers help us in determining the location (map position) of genes that control important traits.
• Types of molecular markers are: restriction fragment length polymorphisms (RFLPs); randomly amplified polymorphic DNAs (RAPDs); variable number of tandom repeats (VNTRs).
Recognition or Cloning sites
• The vector must also have at least one unique restriction endonuclease recognition site, to enable foreign DNA to be inserted into the vector, for the generation of a recombinant DNA molecule.
• Presence of a unique restriction site allows the particular restriction enzyme to cut the vector only at that site.
• Most of the commonly used vectors contain unique recognition sites for several restriction enzymes in a small region of DNA, which is referred to as a polylinker or multiple cloning site (MCS).
• A polylinker provides flexibility in the choice of restriction enzyme(s), that can be used for cloning.
• The ligation of alien DNA is carried out at a restriction site present in one of the two antibiotic resistance genes.
• Selection of recombinants, due to inactivation of antibiotics, is a burdensome procedure, because it requires simultaneous plating on two plates having different antibiotics.
• Alternative selectable markers have been developed which differentiate recombinants from non recombinants on the basis of their ability to produce colour in the presence of chromogenic substrate.
• In this, a recombinant DNA is inserted within the coding sequence of an enzyme, 0-galactosidase. This results in the inactivation of the enzyme, which is referred to as insertional inactivation.
• The presence of a chromogenic substrate gives blue coloured colonies, if the plasmid in the bacteria does not have an insert. Presence of an insert results in insertional inactivation of the p-galactosidase, and the colonies do not produce any colour, which are identified as recombinant colonies.
• The presence of restriction sites within the markers fefR and ampR permits an easy selection for cells transformed with the recombinant pBR322. Insertion of the DNA fragment into the plasmid using restriction enzyme, Pstl or Pvul, places the DNA insert within the gene ampR and thus makes it nonfunctional.
• Bacterial cells containing such a recombinant pBR322 will be unable to grow in the presence of ampicillin, but will grow on tetracycline containing medium. Similarly, when restriction enzyme BamHI or Sail is used, the DNA insert is placed within the gene fefR making it nonfunctional.
• The inactivation of a gene by insertion of a DNA sequence (in this case, the DNA insert) within it is known as insertional inactivation. Bacterial cells possessing such a recombinant pBR322 will, therefore, grow on ampicillin but not on tetracycline. The feature of insertional inactivation allows an easy selection of a single bacterial cell having recombinant pBR322 from among 10s other types of cells.[/userpro_private]