3-1.1 Introduction:

Nucleic acid molecules like deoxyribonucleic acids (DNA), ribonucleic acids (RNA) are basic, essential and primary molecules for all molecular biology related research. Before discussing the common methods to detect nucleic acids let us we learn about the isolation of pure form of nucleic acid.

3-1.2 Preparation Step:

First important step is to establish if a given sample contains DNA or RNA and whether it is in pure form, since many samples will contain both species as well as other contaminants such as cellular proteins. Irrespective of the method applied for detection, the optimal nucleic acid isolation protocol must provide

a) Reproducible result

b) No degradation of the sample and

c) Safer handling.

In deciding what method of nucleic acid measurement is appropriate, three issues are critical: specificity, sensitivity, and interfering substances. Based on these features different detection techniques are to be followed.

3-1.3 Traditional Detection Methods:

3-1.3.1 UV spectroscopy

Majority of bio-molecules intrinsically absorb light in the ultraviolet and not in the visible range. This property of UV absorbance can be used to quickly estimate the concentration and purity of DNA and RNA (also proteins) in a analytical sample. The amount of DNA in a sample can be estimated by looking at its absorbance at a wavelength of 260nm or 280nm (in the UV region). Purines and pyrimidines have absorbance maxima slightly below and above 260 respectively. Thus the absorbance maxima of different fragments of DNA vary somewhat depending on their subunit composition. Contaminants like proteins exhibit two absorbance peaks, one between 215-230 nm (due to peptide bonds absorption) and at about 280 nm (absorption by aromatic amino acids-tyrosine, tryptophan and phenylalanine). Remember that although proteins have little absorbance at 260 nm, both proteins and nucleic acids absorb light at 280 nm. That is the reason why, if nucleic acids and proteins are mixed in the same sample, their spectra interfere (overlap) with one another.