Gene cloning/ Recombinant DNA technology / Genetic engineering:

• Integration of specific fragment of foreign DNA into a cell through a suitable vector in such a way that the inserted DNA replicate independently and transferred to progenies as a result of cell division.
• Recombinant DNA molecule is a vector into which the desired DNA fragment has been inserted.
• Recombinant DNA molecule is produced by joining together two or more DNA segments usually originated from different organisms.

Steps in gene cloning:

1. Identification and isolation of the desired gene or DNA fragment to be cloned

(Restriction digestion and electrophoresis)

2. Insertion of the isolated gene in a suitable vector (ligation)
3. Introduction of this vector into a suitable organism or cell called host (transformation)
4. Selection of transformed host cells (selectable markers)
5. Multiplication / integration followed by expression of the introduced gene in the host.

Enzymes involved:Restriction endonucleases, DNA ligases, DNA polymerases, RNA polymerases and reverse transcriptases.

Vectors used in gene cloning: Tumor inducing (Ti) plasmid of Agrobacterium tumefaciens, pBR322, Bacteriophages, cosmid vectors (derived from phage λ).

Ti plasmid of Agrobacterium tumefaciens:

• Ti plasmid has a T-DNA region (15-24 kb) which is bounded by a pair of 24 bp repeats. T-DNA carries genes for auxin, cytokinins and opine synthesis which are responsible for tumor formation.
• Transfer of T-DNA depends on 35 kb virulence (vir) region of the Ti plasmid.
• The protein products of these genes respond to phenolics to generate a copy of T-DNA and mediate its transfer into the cell.
• The T-DNA when transferred from the Agrobacterium to the plant cell integrates with the chromosome, and the plant cells which are affected begin to synthesize opines, auxins and cytokinins.
• Plant wound exudates contain phenolics, which attract Agrobacterium and induce virgenes.
• The exogenous DNA is inserted into the T-DNA region of the Ti plasmid by

 homologous recombination using an intermediate vector system or directly using binary vectors.