ABSTRACT
Southern1 rst described the detection of speci c nucleotide sequences from a pool of digested DNA fragments separated by electrophoresis and transferred onto a solid support.2 It was thus termed the Southern blot. In conjunction with lter hybridization, the method relied on the complimentary nature of the double-stranded DNA and its reversible denaturation/renaturation 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 modi-ed 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 (see Chapter 11) and protein accumulation (see Chapter 16). To improve throughput, DNA samples can be spotted, either manually or by using commercially available devices, onto membranes directly before hybridization without gel electrophoresis. This modi cation 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 modi ed for high throughput and global analysis of gene expression pro les. Although the use of Southern hybridization has declined with the advent of polymerase chain reaction (PCR) and real-time PCR, it remains an essential technique for gene cloning, library screening, or DNA mapping for species without a sequenced genome.2-6
5.1 Preparation of DNA Samples ................................................................................................. 78 5.1.1 Restriction Enzyme Digestion of Genomic DNA....................................................... 78 5.1.2 Agarose Gel Electrophoresis of DNA ........................................................................ 78
5.2 Blotting DNA onto Nylon Membranes ...................................................................................80 5.2.1 Capillary Transfer Method ......................................................................................... 81 5.2.2 Alkaline Transfer Method .......................................................................................... 83 5.2.3 Vacuum Transfer Method ........................................................................................... 83
5.3 Preparation of DNA Dot/Slot Blots ........................................................................................84 5.4 Hybridization Procedures .......................................................................................................85
