conventionally been labeled with radioactive isotopes, but attention is increasingly being given to nonisotopic labeling. When isotopically labeled probes are used, the pattern of probe binding is visualized with autoradiography (see examples in Box 1-A and Figure 1-5).
The complete process—DNA digestion, electrophoresis, membrane transfer, and hybridization—was developed by Edwin Southern in 1975;14 in its present modified form, it is still usually referred to as Southern blotting. These procedures are routinely used in molecular biology, biochemistry, genetics, and clinical DNA diagnosis; there is no difference in their forensic application. Differences among individuals are expressed as differences in the lengths of RFs.15 RFLPs can result from several kinds of differences at the level of the genome:
Mutations that alter the base sequence at a restriction-enzyme recognition, or cleavage, site can result in a loss of the cutting site or the generation of a cutting site that was not present before. Insertion or deletion of nucleotides between two cleavage sites also changes RF lengths. Variation of these sorts is generally associated with a small number of alleles. For example, the loss or gain of a particular cleavage site might be responsible for only two alleles.
Some regions of DNA contain multiple segments of short-sequence repeats. Consequently, there is a class of RFs that differ in the number of repeated segments present. Some VNTR polymorphisms have a small number of alleles, and the patterns of RFs that represent each of the alleles at a given locus can be readily distinguished. But highly polymorphic VNTR loci have 50-100 alleles or even more. In that situation, the distribution of RF size is essentially continuous; alleles with RFs close in size might not be resolvable with electrophoresis, and the limit of resolution must be defined operationally. Because of the extensive variability, the VNTR class of RFLPs has proved the most informative in distinguishing among persons.
RFLP analysis with single-locus probes is usually designed to result in a simple pattern of one or two RFLP bands, depending on whether the