Cleavase Fragment Length Polymorphism Analysis for Genotyping and Mutation Detection

DNA sequencing is the gold standard in DNA diagnostics and is the only absolute means of establishing the identity of a new mutation. However, the clinical cost of obtaining this information is often prohibitive, particularly when large DNA fragments are interrogated for the presence of any of a number of either known or previously undescribed genetic alterations (1 ). Instead, several mutation scanning methods have been developed to eliminate the need to sequence every nucleotide when it is only the precise identity of one or a few nucleotides that is clinically significant. Until now such methods have provided only a “yes” or “no” answer in determining whether a test sample differs from a known reference. Relatively few methods have been proven capable of unambiguously identifying unique nucleic acid variants, particularly when multiple sequence changes occur (2 ). Consequently, the majority of existing mutation scanning methods are unsuitable for PCR-based genotyping applications in which regions of sequence variability are used to categorize isolates for their similarities to known variants.