ABSTRACT
Contents Southern1 first described the detection of specific nucleotide sequences from a pool of digested deoxyribonucleic acid (DNA) fragments separated by electrophoresis and transferred onto a solid support.2 It was thus termed the Southern blot. In conjunction with filter hybridization, the method relied on the complementary nature of the doublestrand DNA and its reversible denaturation/rena-turation properties to detect the presence and abundance of target DNA sequences in the fractionated DNA population, using a labeled probe of the DNA of interest. Although the exact procedure has since been modified extensively to achieve a higher degree of sensitivity, the principles remain the same and have had a profound impact on modern molecular biology. With changes, similar procedures are being used to detect levels of gene expression (so-called Northern blot hybridization, see Chapter 6) and protein accumulation (so-called Western blot hybridization, see Chapter 8). To 0-8493-0815-1/04/$0.00+$ 1.50
improve throughput, DNA samples can be spotted, either manually or using commercially available devices, onto membranes directly before hybridization without gel electrophoresis. This modification is termed dot/slot blot hybridization. Unlike Southern blot analysis, where hybridization signals can be resolved according to the sizes of the hybridizing DNA, dot/slot blot analysis is most commonly used for comparison of the “total” signal level among individual samples. It was the forerunner of the present day’s DNA microarray technology, which was further modified for high-throughput and global analysis of gene-expression profiles (see Chapter 15). Today, Southern hybridization remains as a fundamental method for gene cloning, library screening, DNA mapping, and confirmation of transgene integration.2-6
I. Preparation of DNA Samples
A typical Southern blot hybridization involves digestion of the DNA (usually genomic DNA) with appropriate restriction enzyme(s) and fractionation of the digested DNA fragments by agarose gel electrophoresis. The gel containing fractionated DNA is then blotted onto a nylon membrane and immobilized prior to hybridization. For genomic Southern hybridization, only high-molecularweight genomic DNA should be used.
