Over-view of cloning: Cloning refers to the process of producing genetically identical organism. In general, for biotechnology related applications, cloning is used to produce DNA, either as a part of a functional gene or part of regulatory region such as promoter. An outline of basic steps involved in cloning is given in Figure 13.1. It has multiple steps to achieve cloned gene in a vector for amplification.

1. Isolation or amplification of gene fragment from genome of the organism.

2. Restriction digestion of gene fragment and vector

3. Ligation of cut gene product and vector

4. Insertion of ligated DNA or recombinant DNA into the host.

5. Screening and selection of cells containing recombinant DNA.

A complete procedure of cloning involves usage of multiple enzyme or molecular tools to perform these steps. Before discussing minor details of these procedure, we will discuss the enzymes involved in cloning procedure and their enzymatic mechanism. Besides this we will also discuss about molecular accessories to facilitate cloning in special conditions.

Molecular Accessories Developed for cloning

1. Linker Molecule-An amplified foreign DNA may have restriction enzymes at their terminus to give cohesive end to facilitate ligation into the vector. But in cases when foreign DNA is a genome product and there is least possibility to keep restriction enzymes at the ends. Cloning of these fragments is facilitated by the help of a linker molecules. Linker molecules are the short double stranded DNA (8-10 nucleotide long) and has restriction sites at their ends. Forex, a typical linker molecule with EcoRI site is shown in Figure 13.2. Linker molecule is incubated with foreign DNA and ligated by the action of T4 DNA ligase to generate chimeric DNA. The chimeric DNA is digested with EcoRI to generate cohesive ends. It is now incubated with EcoRI digested vector in the presence of DNA ligase to get circular clone (Figure 13.2).