This method, named after its inventor, Edward Southern, involves the following steps (Fig. 34.13). First, DNA is extracted from cells, for example, a patient’s WBC. Second, the DNA is cleaved into many fragments using a restriction enzyme. Third, the resulting fragments (all of which are negatively charged) are separated on the basis of size by electrophoresis. [Note: Because the large fragments move more slowly than the smaller ones, the lengths of the fragments, usually expressed as the number of base pairs, can be calculated from comparison of the position of the band relative to standard fragments of known size.] The DNA fragments in the gel are denatured and transferred (blotted) to a nitrocellulose membrane for analysis. If the original DNA represents the individual’s entire genome, the enzymic digest contains ≥106 fragments. The gene of interest is on only one (or a few if the gene itself was fragmented) of these pieces of DNA. If all the DNA fragments were visualized by a nonspecific technique, they would appear as an unresolved blur of overlapping bands. To avoid this, the last step in Southern blotting uses a probe to identify the DNA fragments of interest. The patterns observed on Southern blot analysis depend both on the specific restriction endonuclease and on the probe used to visualize the restriction fragments. [Note: Variants of the Southern blot have been facetiously named northern if RNA is being studied (see p. 499) and western if protein is being studied (see p. 500), neither of which relates to anyone’s name or to points of the compass.]
B. Mutation detection
Southern blotting can detect DNA mutations such as large insertions or deletions, trinucleotide repeat expansions, and rearrangements of nucleotides. It can also detect point mutations (replacement of one nucleotide by another; see p. 449) that cause the loss or gain of restriction sites. Such mutations cause the pattern of bands to differ from those seen with a normal gene. Longer fragments are generated if a restriction site is lost. For example, in Figure 34.13, person 2 lacks a restriction site present in person 1. Alternatively, the point mutation may create a new cleavage site with the production of shorter fragments. [Note: Most sequence differences at restriction sites are harmless variations in the DNA.]
VI. RESTRICTION FRAGMENT LENGTH POLYMORPHISM
It has been estimated that the genomes of any two unrelated people are 99.5% identical. With 6 billion bp in the diploid human genome, that represents variation in ~30 million bp. These genome variations are the result of mutations that lead to polymorphisms. A polymorphism is a change in genotype that can result in no change in phenotype or a change in phenotype that is harmless, causes increased susceptibility to a disease, or, rarely, causes the disease. It is traditionally defined as a sequence variation at a given locus (allele) in >1% of a population. Polymorphisms primarily occur in the 98% of the genome that does not encode proteins (that is, in introns and intergenic regions). A restriction fragment length polymorphism (RFLP) is a genetic variant that can be observed by cleaving the DNA into fragments (restriction fragments) with a restriction endonuclease. The length of the restriction fragments is altered if the variant alters the DNA so as to create or abolish a restriction site. RFLP can be used to detect human genetic variations, for example, in prospective parents or in fetal tissue.
A. DNA variations resulting in RFLP
Two types of DNA variations commonly result in RFLP: single-base changes in the DNA sequence and tandem repeats of DNA sequences.