Enzymatic and Chemical Cleavage Methods to Identify Genetic Variation

Publisher Summary This chapter discusses two high sensitivity mutation detection methods to identify genetic variation: chemical and enzymatic cleavage of mismatch. Although there are several methods for high sensitivity detection of known mutations, unknown mutations are more difficult to uncover. However, methods for the latter have improved significantly and are utilized in practical applications. These include the establishment of a single nucleotide polymorphism (SNP) map for a specific part of the genome in a specific animal population or the screening for unknown mutations in important genes, such as cancer susceptibility genes. These genes are large, with many exons, and thus hundreds of possible mutations that affect the functions of those proteins are found. The mutation detection methods serve as tools for reverse genetics to screen for chemically induced point mutations in specific regions of specific genes and for specific mutations in the genome of emerging pathogenic microorganisms. In mutation screening methods, the mutated DNA helix is first converted to mismatch heteroduplexes when the polymerase chain reaction products of two alleles of the gene of interest are amplified, mixed, denatured, and rehybridized. Next, the chemical or enzymatic properties of a mismatched base are exploited to lead to a break in the DNA strand near the mismatch. Finally, a suitable fragment analysis method is used to visualize the shortened DNA fragments that are produced by the DNA break in either single-stranded or double-stranded form.