Cloning Vectors for Recombinant DNA

One of the important uses of recombinant DNA technology is the cloning of (i) random DNA or cDNA segments, often used as probes or (ii) specific genes, which may be either isolated from the genome or synthesized organochemically or in the form of cDNA from mRNA. This cloning of DNA is possible only with the help of another DNA molecule, which is capable of replicating in a host. This other DNA molecule is often used in the form of a vector, which could be a plasmid, a bacteriophage, a derived cosmid or phagemid, a transposon or even a virus. Techniques should also be available, which will allow selection of chimeric genomes obtained after insertion of foreign DNA from a mixture of chimeric and the original vector. Another critical desired feature of any cloning vector is that it should possess a site at which foreign DNA can be inserted without disrupting any essential function. Therefore, in each case an enzyme will also have to be selected which will cause a single break. Sometimes vectors ate modified by inserting a DNA segment to create unique site(s) for one or more enzymes to facilitate its use in gene cloning. This DNA with restriction sites for several enzymes is sometimes called a polylinker.

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