Single-Base-Pair Discrimination of Terminal Mismatches by Using Oligonucleotide Microarrays and Neural Network Analyses

ABSTRACT The effects of single-base-pair near-terminal and terminal mismatches on the dissociation temperature ( T d ) and signal intensity of short DNA duplexes were determined by using oligonucleotide microarrays and neural network (NN) analyses. Two perfect-match probes and 29 probes having a single-base-pair mismatch at positions 1 to 5 from the 5′ terminus of the probe were designed to target one of two short sequences representing 16S rRNA. Nonequilibrium dissociation rates (i.e., melting profiles) of all probe-target duplexes were determined simultaneously. Analysis of variance revealed that position of the mismatch, type of mismatch, and formamide concentration significantly affected the T d and signal intensity. Increasing the concentration of formamide in the washing buffer decreased the T d and signal intensity, and it decreased the variability of the signal. Although T d s of probe-target duplexes with mismatches in the first or second position were not significantly different from one another, duplexes with mismatches in the third to fifth positions had significantly lower T d s than those with mismatches in the first or second position. The trained NNs predicted the T d with high accuracies ( R 2 = 0.93). However, the NNs predicted the signal intensity only moderately accurately ( R 2 = 0.67), presumably due to increased noise in the signal intensity at low formamide concentrations. Sensitivity analysis revealed that the concentration of formamide explained most (75%) of the variability in T d s, followed by position of the mismatch (19%) and type of mismatch (6%). The results suggest that position of the mismatch at or near the 5′ terminus plays a greater role in determining the T d and signal intensity of duplexes than the type of mismatch.