Determinants of sensitivity and specificity in spotted DNA microarrays with unmodified oligonucleotides

Abstract

DNA microarrays with unmodified oligonucleotides are a cost-effective alternative to cDNA microarrays. This study examined how purity, length, homology and GC content of the oligonucleotide probes influence the sensitivity and specificity of the method using cyanobacterial genes. Oligonucleotide purification by high pressure liquid chromatography was omitted without significant reduction in hybridization sensitivity. For two of three genes tested, a reduction in oligonucleotide length did not reduce hybridization sensitivity, and maximum sensitivity was achieved with probes that were 45 nt long. Oligonucleotide probes with ≤71% contiguous sequence identity to non-target DNA cross-hybridized with the sequences at a rate of < 8%. Cross-hybridization decreased as the GC content decreased from 65% to 55% or 35%. These results support the following criteria for selecting unmodified oligonucleotide probes that generate sensitive and specific microarrays: length of 45 nt, ≤71% identity to non-target sequences, and ≤55% GC content. In most of the bacterial species tested, oligonucleotide probes meeting these criteria were successfully designed for more than 95% of genes.