Optimization of Single-Base-Pair Mismatch Discrimination in Oligonucleotide Microarrays

Abstract

The discrimination between perfect-match and single-base-pair-mismatched nucleic acid duplexes was investigated by using oligonucleotide DNA microarrays and nonequilibrium dissociation rates (melting profiles). DNA and RNA versions of two synthetic targets corresponding to the 16S rRNA sequences of Staphylococcus epidermidis (38 nucleotides) and Nitrosomonas eutropha (39 nucleotides) were hybridized to perfectmatch probes (18-mer and 19-mer) and to a set of probes having all possible single-base-pair mismatches. The melting profiles of all probe-target duplexes were determined in parallel by using an imposed temperature step gradient. We derived an optimum wash temperature for each probe and target by using a simple formula to calculate a discrimination index for each temperature of the step gradient. This optimum corresponded to the output of an independent analysis using a customized neural network program. These results together provide an experimental and analytical framework for optimizing mismatch discrimination among all probes on a DNA microarray.DNA microarray technology provides parallel nucleic acid hybridizations for a large number of immobilized oligonucleotides or larger DNA fragments on a small surface area (21). In clinical and environmental microbiology, this technology has been used for assessing gene expression (19), characterizing whole genomes (5), identifying bacteria (8, 10, 28), and monitoring microbial populations (12,22). We anticipate that, in the next several years, the application of DNA microarrays to environmental microbiology will greatly improve the understanding of complex microbial communities, which are typically composed of many microbial species.In general, oligonucleotide DNA microarrays containing 15-to 25-mer oligonucleotide probes provide greater discrimination than microarrays composed of larger PCR-amplified DNA fragments. However, a central challenge to the application of DNA microarrays in environmental microbiology is achieving the specificity needed to resolve complex microbial populations, including discriminating between target and nontarget populations that differ by a single nucleotide (10). This level of specificity is needed to resolve variants of highly conserved genes (e.g., those encoding the rRNAs) and to distinguish between closely related target and nontarget microorganisms.In conventional hybridization assays, single-base-pair discrimination is achieved by adjusting the hybridization conditions (e.g., temperature, ionic strength, or formamide concentration) or washing conditions (dissociation) of the probe-target duplex (31). In DNA microarray assays, however, this approach is difficult to use since one set of hybridization and wash conditions does not provide optimal target discrimination for all probes on the microarray. We therefore have developed an alternative approach that uses differences in thermal dissociation rates of probe-target duplexes to resolve matched and mismatched probe-target duplexes (13,25).The oligonucleotide DNA microarray used ...

Introduction to Microarray‐Based Detection Methods

“…It has been demonstrated that the hybridization intensity of MM probes can depend on the nucleotide type (i.e. A, C, G or T) and position of the MM relative to the termini [178,179], and that some MM probes yield even higher signal intensities with the target than those of corresponding PM probes [125]. Defined mismatches are more difficult to detect (e.g.…”

“…GG or GT), as well as mismatches that reside in a context (GC-rich domain). Even well-designed probes can display differences in maximal hybridization capacity of 2 orders of magnitude under different hybridization conditions [22] and thus it is difficult to find one set of conditions that is optimal for all probes on an array [85,178]. Factors affecting duplex formation on DNA microarrays include probe density, microarray surface composition, spacer length and the stabilities of oligonucleotidetarget duplexes, intra-and intermolecular self-structures, and RNA secondary structures [75,121,133].…”

Application of a Microarray-Based Assay for the Study of Genetic Diversity of West Nile Virus

“…The strongest signal ratios between perfect matched and mismatched sequences were observed when mutations were located near the center of an oligoprobe, and the shortest probes always had better discriminatory power (Urakawa et al, 2003). Although the microarray hybridization method provides limited information about the position of mutations in the analyzed genomic region when compared to sequencing, it has the significant advantage of allowing the identification of "hot spots" where random mutations occur within short (a few nucleotides) areas.…”

“…The efficiency of microarrays for identification and discrimination of closely related bacteria and viruses has been previously demonstrated Hsia et al, 2007;Laassri et al, 2003;Nordström et al, 2005;Volokhov et al, 2002;Wade et al, 2004). The use of oligonucleotide microchips for screening of random mutations is based on the ability of microarrays to identify the presence of singlenucleotide mutations in the hybridization template (Hacia et al, 1999;Urakawa et al, 2003).…”

Marine microbial ecology in a molecular world: what does the future hold?

“…A molecular taxonomy will also enable the development of macro/microarrays on which a large component of the microbial community can be assayed simultaneously. Such 'phylochips' will be instrumental for ecological studies of microorganisms and for rapidly establishing the presence or absence of species of interest Urakawa et al, 2003).…”