Influence of Dangling Ends and Surface-Proximal Tails of Targets on Probe-Target Duplex Formation in 16S rRNA Gene-Based Diagnostic Arrays

Abstract

Dangling ends and surface-proximal tails of gene targets influence probe-target duplex formation and affect the signal intensity of probes on diagnostic microarrays. This phenomenon was evaluated using an oligonucleotide microarray containing 18-mer probes corresponding to the 16S rRNA genes of 10 waterborne pathogens and a number of synthetic and PCR-amplified gene targets. Signal intensities for Klenow/random primer-labeled 16S rRNA gene targets were dissimilar from those for 45-mer synthetic targets for nearly 73% of the probes tested. Klenow/random primer-labeled targets resulted in an interaction with a complex mixture of 16S rRNA genes (used as the background) 3.7 times higher than the interaction of 45-mer targets with the same mixture. A 7-base-long dangling end sequence with perfect homology to another single-stranded background DNA sequence was sufficient to produce a cross-hybridization signal that was as strong as the signal obtained by the probe-target duplex itself. Gibbs free energy between the target and a well-defined background was found to be a better indicator of hybridization signal intensity than the sequence or length of the dangling end alone. The dangling end (Gibbs free energy of ؊7.6 kcal/mol) was found to be significantly more prone to target-background interaction than the surface-proximal tail (Gibbs free energy of ؊64.5 kcal/mol). This study underlines the need for careful target preparation and evaluation of signal intensities for diagnostic arrays using 16S rRNA and other gene targets due to the potential for target interaction with a complex background.

In Situ-Synthesized Virulence and Marker Gene Biochip for Detection of Bacterial Pathogens in Water

“…2b). Extensive unevenness in signal intensity among probes for a given target is well documented (5,44,46,56). This variability may be attributed to various factors, including probe and target secondary structure (9, 27, 47), target length (33), ⌬G°d uplex (23), and even the position of fluorescent labels (68).…”

“…Raw spot intensities were divided by the mean signal intensity of 22 empty control spots to obtain signal-to-noise ratios (SNRs) (56). The SNR was computed for each wash step between 30 and 45°C, and the values were subsequently averaged.…”

“…Biochip hybridization and melting curve development. Hybridization and melting profile generation were performed using previously established protocols (56,64). The method used for melting profile analysis was based on methods used in previous studies utilizing this approach for microarrays using 16S rRNA targets (15,29,34,60).…”

“…The probes were attached to the substrate via a spacer consisting of Ts and C 18 with an effective length of 12 nucleotides. The biochip also contained 22 randomly spaced control spots containing solely linker chemistry to assess background signal intensity (56,64 Tap water (40 liters), river water (10 liters; Red Cedar River, East Lansing, MI), and tertiary effluent (20 liters; wastewater treatment plant in East Lansing, MI) were sampled and filtered through 0.45-m nitrocellulose filters (Millipore, Billerica, MA). gDNA was extracted from the filters using a MegaPrep UltraClean soil DNA kit (Mo Bio Laboratories, Carlsbad, CA) according to the manufacturer's instructions.…”

“…PCR products pooled from different PCRs were labeled using an aminoallyl dUTP (aa-dUTP) incorporation and cyanine dye coupling protocol as described previously (56,64), with minor modifications. Briefly, PCR products were amplified, and aa-dUTP was incorporated into the amplification products with a Bioprime DNA labeling kit (Invitrogen, San Diego, CA) using a ratio of aa-dUTP to dTTP of 5:1 and an incubation time of 120 min.…”