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Evaluating single‐base‐pair discriminating capability of planar oligonucleotide microchips using a non‐equilibrium dissociation approach
Author(s) -
Li Emily Sze Ying,
Ng Johnson Kian Kok,
Wu JerHorng,
Liu WenTso
Publication year - 2004
Publication title -
environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.954
H-Index - 188
eISSN - 1462-2920
pISSN - 1462-2912
DOI - 10.1111/j.1462-2920.2004.00648.x
Subject(s) - oligonucleotide , dissociation (chemistry) , planar , biology , base pair , biological system , analytical chemistry (journal) , materials science , chromatography , gene , computer science , genetics , chemistry , computer graphics (images)
Summary The capability of planar rRNA‐based oligonucleotide microarrays for single‐base‐pair discrimination was evaluated using an approach that compares the non‐equilibrium dissociation profiles and dissociation temperatures (T d s) of all probe‐target duplexes simultaneously. Three sets of 16S rRNA gene specific probes at different levels of specificity were used along with their counter probes for individual sets having either one or two mismatches (MM) to their targets at specific external (next to terminus) and various internal positions. Criteria based on the T d approach and a discrimination index (DI) were proven to be competent in discriminating PM from internal MM duplexes, but not always for external MM duplexes. Maximal DI for separating PM duplexes from ones with two and one internal MM usually occurred at temperatures approximately 5–10°C and 10–15°C, respectively, higher than the T d s of the PM duplexes. Washing buffer type and salt concentration, and MM number and position were shown statistically to affect dissociation profiles, T d , and single‐base‐pair discriminating capability. The reusability potential of the planar microchip was further demonstrated.