CLASSIFICATION AND IDENTIFICATION OF FUNGALSEQUENCES USING CHARACTERISTIC RESTRICTIONENDONUCLEASE CUT ORDER

摘要

Restriction Fragment Length Polymorphism (RFLP) is a powerful molecular tool that isextensively used in the molecular fingerprinting and epidemiological studies of microor-ganisms. In a wet-lab setting, the DNA is cut with one or more restriction enzymes andsubjected to gel electrophoresis to obtain signature fragment patterns, which is utilizedin the classification and identification of organisms. This wet-lab approach may not bepractical when the experimental data set includes a large number of genetic sequencesand a wide pool of restriction enzymes to choose from. In this study, we introduce anovel concept of Enzyme Cut Order — a biological property-based characteristic ofDNA sequences which can be defined and analyzed computationally without any align-ment algorithm. In this alignment-free approach, a similarity matrix is developed basedon the pairwise Longest Common Subsequences (LCS) of the Enzyme Cut Orders. Thechoice of an ideal set of restriction enzymes used for analysis is augmented by usinggenetic algorithms. The results obtained from this approach using internal transcribedspacer regions of rDNA from fungi as the target sequence show that the phylogenetically-related organisms form a single cluster and successful grouping of phylogenetically closeor distant organisms is dependent on the choice of restriction enzymes used in the anal-ysis. Additionally, comparison of trees obtained with this alignment-free and the legacymethod revealed highly similar tree topologies. This novel alignment-free method, whichutilizes the Enzyme Cut Order and restriction enzyme profile, is a reliable alternativeto local or global alignment-based classification and identification of organisms.