4-1.1. Introduction

Every gene manipulation procedure requires genetic materials like DNA and RNA. Nucleic acids occur naturally in association with proteins and lipoprotein organelles. The dissociation of a nucleoprotein into nucleic acid and protein moieties and their subsequent separation, are the essential steps in the isolation of all species of nucleic acids. Isolation of nucleic acids is followed by quantitation of nucleic acids generally done by either spectrophotometric or by using fluorescent dyes to determine the average concentrations and purity of DNA or RNA present in a mixture. Isolating the genetic material (DNA) from cells (bacterial, viral, plant or animal) involves three basic steps-

•  Rupturing of cell membrane to release the cellular components and DNA

•  Separation of the nucleic acids from other cellular components

•  Purification of nucleic acids

4-1.2. Isolation and Purification of Genomic DNA

Genomic DNA is found in the nucleus of all living cells with the structure of double-stranded DNA remaining unchanged (helical ribbon). The isolation of genomic DNA differs in animals and plant cells. DNA isolation from plant cells is difficult due to the presence of cell wall, as compared to animal cells. The amount and purity of extracted DNA depends on the nature of the cell.

The method of isolation of genomic DNA from a bacterium comprises following steps (Figure 4-1.2.)-

1.  Bacterial culture growth and harvest.

2.  Cell wall rupture and cell extract preparation.

3.  DNA Purification from the cell extract.

4.  Concentration of DNA solution.