The following steps are involved in the method known as Southern blotting, named after its inventor Edward Southern (Fig. 34.13). Initially, DNA is extracted from cells, such as a patient's white blood cells (WBC). Next, the DNA is fragmented using a restriction enzyme. The resulting fragments, which are negatively charged, are then separated based on size through electrophoresis. By comparing the position of the fragments to standard fragments of known size, the lengths of the fragments can be determined. The DNA fragments in the gel are denatured and transferred to a nitrocellulose membrane for analysis. If the original DNA represents the entire genome of an individual, the enzymic digest contains over one million fragments. The gene of interest is located on one or a few of these DNA fragments. To avoid visualizing all the DNA fragments as a blurry overlap, a probe is used in the last step of Southern blotting to identify the DNA fragments of interest. The patterns observed in Southern blot analysis depend on both the specific restriction endonuclease used and the probe utilized to visualize the restriction fragments.

Southern blotting is capable of detecting various DNA mutations, including large insertions or deletions, trinucleotide repeat expansions, rearrangements of nucleotides, and point mutations. Point mutations result in the loss or gain of restriction sites, altering the pattern of bands compared to a normal gene. If a restriction site is lost, longer fragments are generated. Conversely, a point mutation may create a new cleavage site, leading to the production of shorter fragments.

The genomes of unrelated individuals are estimated to be 99.5% identical. With 6 billion base pairs in the diploid human genome, this represents a variation of approximately 30 million base pairs. These variations in the genome are the result of mutations that give rise to polymorphisms. A polymorphism refers to a change in genotype that may or may not result in a change in phenotype. It can be a harmless variation, increase susceptibility to a disease, or rarely, cause the disease itself. Polymorphisms are typically defined as sequence variations at a specific locus (allele) that are present in more than 1% of a population. They primarily occur in the non-protein coding regions of the genome (introns and intergenic regions). A restriction fragment length polymorphism (RFLP) is a genetic variant that can be observed by fragmenting the DNA with a restriction endonuclease. If the variant alters the DNA sequence to create or abolish a restriction site, the length of the restriction fragments will be affected. RFLP can be used to detect genetic variations in humans, for instance, in prospective parents or fetal tissue.

There are two types of DNA variations that commonly result in RFLP: single-base changes in the DNA sequence and tandem repeats of DNA sequences.
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.