Desirable Properties of Plasmid Cloning Vehicles

Desirable Properties of Plasmid Cloning Vehicles
An ideal cloning vehicle would have the following three properties:
(I) Low molecular weiglu;
(2) Ability to confer readily selectable phenotypic traits on host cells;
(3) Single sites for a large number of restriction endonucleases, preferably in genes with a readily scorable phenotype.
The advantages of a low mol. wt. arc scvcralfold. Firstly, the plasmid is much easier to handle, i.e. it is more resistant to damage by shearing. and is readily isolated from host cells. Secondly, low mol. wt. plasmids are
usually present as multiple copies (Table 3.2) and this not only facilitates their isolation but leads to gene dosage effects for all cloned genes. Finally. with a low mol. wt. there is less chance that the vector will have multiple substrate sites for any restriction endonuclease (see below). After a piece of foreign DNA is inserted into a vector (lie resulting chimaeric molecules have to be transformed into a suitable recipient. Since the efficiency of transformation is so low it is essential that the chimacra have some readily scorablc phenotype. Usually this rcsults from some gene, e.g. antibiotic resistance, carried on the vector but could also be
produced by a gene carried on the inserted DNA. One of the first steps in cloning is to cut the vector DNA and the DNA to be inserted with the same endonuclcasc. If the vector has more than one site for the endonuclease then more than one fragment will be produced. When the two samples of cleaved DNA are subsequently mixed and ligated the resulting chimaeras will, in all probability, lack one of the vector
fragments. It is advantageous if insertion of foreign DNA at endonuclcase. sensitive sites inactivates a gene whose phenotype is readily scorable, for in this way it is possible to distinguish chimaeras from cleaved plasmid molecules which have self.anneaied. Of course, readily detectable insertional inactivation is not essential if the vector and insert are to be joined by the homopolymcr tailing method (see page 21) or if the insert confers a new phenotype on host cclls. Some examples will be presented which illustrate the points raised above but first we shall consider how some of the common plasmids rate as cloning vehicles.