ABSTRACT
Characterization of DNA or the gene plays an important role in molecular biology research. A traditional and powerful tool that is widely used is Southern blot hybridization, which refers to a procedure in which different sizes of DNA molecules are immobilized from agarose gels onto a solid support such as nylon or nitrocellulose membranes and are then hybridized with a labeled DNA probe. In other words, Southern blot refers to DNA-DNA hybridization. This technique was developed by Southern, so named the Southern blot, that has been subjected to later modi„cation.1-2 This blotting method becomes one of the most fundamental and powerful tools used in molecular biology studies. It is a sensitive and reliable method. Its applications are remarkably broad, including general DNA analysis, gene cloning, screening and isolation, DNA mapping, mutant detection, identi„cation of genetic diseases, and DNA ampli„cation.2-5
Based on our extensive experience, detailed protocols are described in this chapter for a successful Southern blot hybridization using nonradioactive or radioactive probes.3-9
The general principles and procedures of Southern blot hybridization are outlined in Figure 7.1. Different DNA molecules are „rst separated based on their molecular weight by standard agarose gel electrophoresis, in which agarose gel serves as a molecular sieve and electrophoresis is the force for migration of negatively charged DNA in the electrical „eld. The separated double-stranded DNA species are denatured into single-stranded DNAs and are then blotted or immobilized onto a nylon or a nitrocellulose membrane. Usually, different positions of DNA molecules in an agarose gel are exactly blotted on their related positions on the membrane. After covalent cross-linking of DNA molecules on the membrane, speci„c band(s) of interest can be then hybridized and detected with a speci„c DNA probe. The nucleotide sequence of one strand of the DNA molecule is complementary to the nucleotide sequence of the other strand. It is exactly the base pairing or complementary rule that makes it possible for a single-stranded DNA probe to hybridize with its target sequences of single-stranded DNA on the membrane.
