Nucleic Acid Blotting Techniques

Abstract

This chapter deals with basic concepts and techniques in nucleic acid blotting. Many of the techniques involved with Southern blotting and Northern blotting are similar. Negatively charged, purified nucleic acids from prokaryotic or eukaryotic cells are separated according to size by electrophoresis through an agarose gel matrix. The RNA or denatured DNA is subse– quently transferred and immobilized onto a membrane com– posed of nitrocellulose or nylon. The nucleic acids on the membrane are then hybridized to a specific labeled “probe,” which consists of homologous single-stranded nucleic acids that carry molecules, allowing detection and visualization of the hybridized probe. Hybridization between the immobilized nucleic acids and labeled probe allows detection of specific DNA or RNA sequences within a complex mixture of DNA or RNA. The specific method of detection and visualization is dependent on the nature of the labeled probe; radioactive probes enable autoradiographic detection, and probes labeled with enzymes facilitate chemiluminescent or colorimetric detection. Nucleic acid blotting yields valuable information pertaining to gene integrity and copy number (Southern blot) and provides a means of analyzing gene expression and mRNA size (Northern blot). These methods can be used to character– ize tissues and cultured cells in the laboratory and often provide valuable information for clinical evaluation of patient samples.