Medical Genetics - V. M. Zaporozhan 2005

Methods for Diagnosing Hereditary Diseases
Molecular-Genetic Methods (DNA Diagnostic Methods)
Other DNA Diagnostic Methods — Use of Restriction Endonucleases

Various molecular diagnostic Methods frequently employ Enzymes known as Restriction Endonucleases (restriction enzymes). They were first discovered in Bacteria. In vivo, these enzymes participate in DNA Repair and the destruction of foreign DNA. Restriction enzymes recognize specific sequences of 4–6, or more rarely 8–12, NUCLEOTIDES in double-stranded DNA and cleave it into fragments at the locations of these sequences, which are referred to as restriction sites. The Number and Length of the resulting DNA Restriction fragments are determined by the frequency of restriction sites and their distribution within the DNA. The more closely spaced the restriction sites are, the shorter the resulting DNA fragments and the greater their total number.

Today, over 500 Different types of bacterial restriction enzymes are known. They are named after the species of microorganism from which they were isolated. Each of these enzymes recognizes its own specific nucleotide sequence. Every restriction enzyme cleaves DNA at strictly defined sites where specific restriction sites are located (Table 10.2).

Table 10.2. Examples of restriction endonucleases and their corresponding restriction sites

The length of the resulting fragments can be analyzed using agarose or Polyacrylamide gel Electrophoresis.

Point Mutations can alter The nucleotide sequence within the restriction site for a specific restriction enzyme. Consequently, such a restriction enzyme will be unable to cleave the mutant DNA fragment (Fig. 10.9). Conversely, in some cases, a mutation creates a new restriction site for a specific enzyme that is normally absent. In both scenarios, the mutant and normal DNA will yield restriction fragments of different lengths, which can be readily detected via electrophoresis (Fig. 10.10).