Abstract
BackgroundThe performance of probes on an oligonucleotide microarray can be characterised in terms of hybridisation signal strength and the ability to discriminate sequence mismatches between the probe and the hybridising target strand, such as those resulting from SNPs. Various properties of the probe affect mismatch discrimination, such as probe length and the position of mismatched bases, and the effects of these factors have been well characterised in a variety of array formats.ResultsA low-density microarray was developed to systematically investigate the effect of a probe’s position within hybridised target PCR products on the tolerance and discrimination of single-nucleotide mismatches between the probe and target. In line with previous reports, hybridisation signals were attenuated by different degrees depending on the identity of the mismatch, the position of the mismatch within the probe, and the length of the PCR product. However, the same mismatch caused different degrees of attenuation depending on the position of the probe within the hybridising product, such that improved mismatch discrimination was observed for PCR products where a greater proportion of the total length was proximal to the array surface.ConclusionsThese results suggest that the degree of mismatch discrimination can be influenced by the choice of PCR primers, providing a means by which array performance could be fine-tuned in addition to manipulation of the properties of the probes themselves.
Highlights
The performance of probes on an oligonucleotide microarray can be characterised in terms of hybridisation signal strength and the ability to discriminate sequence mismatches between the probe and the hybridising target strand, such as those resulting from SNPs
High-density microarrays in a conventional glass slide format with fluorescence detection, such as those used for gene expression studies, pose a relatively high operational burden due to time-consuming protocols and the cost of the equipment required for high-resolution fluorescence detection
PCR primer design PCR primers were designed based on B. pseudomallei sequence to generate PCR products of different lengths and in different positions relative to the probes
Summary
The performance of probes on an oligonucleotide microarray can be characterised in terms of hybridisation signal strength and the ability to discriminate sequence mismatches between the probe and the hybridising target strand, such as those resulting from SNPs. A typical low-density array protocol involves subjecting sample DNA to multiplex PCR with labelled nucleotides or primers, followed by application of the labelled products to the array and identification of the probes to which the products have hybridised. A frequently used approach is to select conserved target genes, enabling amplification using broad-range PCR primers followed by discrimination of different products by hybridisation to specific probes [4]. In order to reliably detect sequence variants (for example, different viral strains or novel viruses) it can be necessary to tolerate sequence differences both in the PCR primers and the probes on the array [5]. In order to detect diverse targets (for example, bacterial and viral pathogens) on a single array, it may be necessary to maximise mismatch discrimination for some targets while tolerating mismatches for others
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