Restriction Enzymes 181probe that hybridizes only to one sequence in a Southern blot experi-
ment, one will find in general only one restriction fragment for a given
allele. During evolution, different alleles can acquire mutations in the
sequences next to the hybridization position that may result in a dif-
ferent length of a particular restriction fragment. This phenomenon is
called a restriction fragment length polymorphism (RFLP) for this
pair of enzyme and probe. Such RFLPs are useful for the identification
of genetic diseases, because the gene defect linked to the RFLP can be
identified in the absence of a phenotypic abnormality or before its
manifestation (188,189). RFLPs can also be used for forensic pur-
poses or to clarify kinship relationships between individuals.4.3. DNA Fingerprints
The pattern of different DNA fragments produced by digesting
genomic DNA with a restriction enzyme can be used to identify not
only the species, but also different strains of a species or even different
individuals, provided an enzyme is used that has many cleavage sites,
such that a pattern rich in information is obtained (190). This approach
has been successfully used for the typing of microorganisms and for
an estimation of the evolutionary relatedness of various species. If the
genomic DNA is too complex, such that individual restriction frag-
ments cannot be resolved as with eukaryotic genomes, one has to label
a subset of all these fragments by hybridization with a DNA probe that
has a sequence complementary to a middle repetitive DNA sequence
in this genome. This procedure results in a pattern of approx 20-50
bands that can be considered as a genetic fingerprint. Genetic finger-
printing begins to form an important part of casework procedure in
forensic science laboratories (191).
4.4. Analysis of the Methylation State of DNA
from Natural Sources
Eukaryotic DNA is methylated to a variable extent on the 5 position
of cytosine, and the subject is discussed in detail in Sections 2.4.1. and
3.1. There is evidence that the methylation state is correlated with the
level of gene expression. Isoschizomer pairs of restriction endonu-
cleases, which allow the analysis of the methylation state of DNA, are
given in Table 7.