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Cloning Vectors

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Cloning Vectors - Lesson Summary

When we link an alien piece of DNA with bacteriophage or plasmid DNA, we can multiply its number to be equal to the copy number of the plasmid or bacteriophage. The first artificial plasmid, which is now commonly used as a cloning vector, is p b r three twenty-two. It was created in nineteen seventy seven and was named after its Mexican creators, Bolivar and Rodriguez. Let us now review the main features that facilitate cloning into a vector such as p b r three twenty-two.  These features are origin of replication, selectable markers and cloning sites. The origin of replication or ori is a sequence of DNA at which replication gets initiated in a plasmid. When a DNA piece is linked to this sequence, it can be made to replicate within the host cells. This sequence also controls the copy number of the linked DNA. Therefore, whenever one has to recover many copies of the target DNA, it should be cloned in a vector whose origin supports a high copy number. The vector also requires a selectable marker, due to which a plasmid is more useful for the cell. Selectable markers help identify and eliminate non-ransformants and exclusively permit the growth of transformants. For example, in E coli, the genes encoding resistance to various antibiotics such as ampicillin, chloramphenicol, tetracycline or kanamycin are used as selectable markers. Another important feature of a cloning vector is a cloning site, which is an artificially constructed region within a vector molecule containing a number of closely spaced recognition sequences. These sequences or recognition sites can recognise several restriction enzymes and therefore the vector can be used to clone a variety of DNA samples. However, the presence of more than one recognition site may lead to complications in gene cloning. In the cloning vector, p b r three twenty-two, the ligation of alien DNA is also carried out at a restriction site present in any one of the two antibiotic resistance genes, which are ampicillin and tetracycline. Selectable markers help identify and eliminate non-transformants and exclusively permit the growth of transformants.   Selectable markers have been developed to differentiate between recombinants and non-recombinants by producing colour in the presence of a chromogenic substance.

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