Plasmids as vectors

Plasmids are defined as autonomous elements, whose genomes exist in the cell as extrachromosomal units. They are self replicating circular (only rarely linear) duplex DNA molecules, which are maintained in a characteristics number of copies in a bacterial cell, yeast cell or even in organelles found in eukaryotic cells. These plasmids can be single copy plasmids that are maintained as one plasmid DNA per cell or multicopy plasmids, which are maintained as 10-20 genomes per cell. There are also plasmids, which are under relaxed replication control, thus permitting their accumulation in very large numbers (up to 1000 copies per cell). These are the plasmids which are used as cloning vectors, due to their increased yield potential.

Circular plasmid DNA which is used as a vector, can be cleaved at one site with the help of a restriction enzyme to give a linear DNA molecule. A foreign DNA segment can now be inserted, by joining the ends of broken circular DNA to the two ends of foreign DNA, thus regenerating a bigger circular DNA molecule that can now be separated by gel electrophoresis on the basis of its size. Selection of chimeric DNA is also facilitated by the resistance genes, which the plasmid may carry against one or more antibiotics. If a plasmid has two such genes conferring resistance against two antibodies and if the foreign DNA insertion site lies within one of these two genes, then the chimeric vector loses resistance against one antibiotic. In such a situation, the parent vector in bacterial cells can be selected by resistance against two antibodies and the chimeric DNA can be selected by retention of resistance against only one of the two antibiotics. 

Authored and Published by;

Raj Abhisek Panda

Recombinant DNA and Gene Cloning

Cloning and Expression Vectors

In recent years, techniques for manipulating prokaryotic as well as eukaryotic DNA have witnessed a remarkable development. This has allowed breakage of a DNA molecule at two desired place to isolate a specific DNA segment and then insert it in another DNA molecule at a desired position. The product thus obtained is called Recombinant DNA and the technique often called genetic engineering. Using, this technique we can isolate and clone single copy of a gene or a DNA segment into an indefinite number of copies, all identical. This become possible because vectors like plasmid and phages reproduce in a host (e.g. E.coli) in their usual manner even after insertion of foreign DNA, so that the inserted DNA will also replicate faithfully with the parent DNA. This technique is called gene cloning. With this technique, genes can be isolated, cloned and characterized, so that the technique has led to significant progress in all areas of molecular biology.

A variety of vectors have been developed which not only allow multiplication, but may also be manipulated in such a way that the inserted gene may express in the host. Due to the importance of a variety of these cloning and expression vectors in genetic engineering experiments, they will discussed in some detail in next posts.  The techniques used for inserting foreign DNA in these vectors and the development of chimeric DNA molecules (for developing molecular probes, gene libraries, etc.) will be discussed in subsequent posts.

Authored and published by

Raj Abhisek Panda

 References;

Elements of BIOTECHNOLOGY, P.K. GUPTA. 2002,Title code no.-BC-22,PP no-14