Effects of Probe Length and Secondary Structure on Dna Microarray Hybridizations: Implications for Design of Pathogen Detection Assays

A number of investigators have proposed using DNA microarrays as part of pathogen detection assays. False negative hybridizations can occur if the target DNA hybridizes to itself and other target DNA, thus inhibiting hybridization to complementary microarray probes. We hypothesized that longer probes can compete with this ‘secondary structure’ better than shorter probes. To test this we constructed a prototype microarray using a series of short and long probes that were specific to the 16S ribosomal DNA gene of Escherichia coli. Three different targets (PCR un-nick translated, PCR nick translated, and nick translated genomic DNA) were hybridized to an array and assessed by a Tyramide Signal Amplification biotin system. Our results verified that hybridization of short probes to target sequences is greatly affected by secondary structure, while long probes provide enhanced signal intensities. Using long probes can offer three specific advantages to microarray design: 1) mitigation of secondary structure, 2) enhanced analytical sensitivity, and 3) greater tolerance to base pair mismatches.