Using a set of 18 deletant strains, we have experimentally validated our cross-hybridization procedure.
Indeed, we observed that reducing the hybridization temperature to 55°C was enough to avoid DNA cross-hybridization and confer specificity to the detection of eGFP transcripts in electroporated embryos (see Figure S1E H).
This result suggests that transcripts from intergenic regions can lead to cross-hybridization, thus validating our concern to examine cross-hybridization of probe with intergenic sequences.
Probes from genomes other than B. cenocepacia were included to allow for cross-hybridization studies focusing on probe specificity and optimization of hybridization conditions.
Thus, in such analyses, quantification of a small difference in signal intensity between PM and MM probes can be difficult due to the presence of cross-hybridization, and thus evaluation of specificity for single nucleotide mismatches is difficult at low target concentrations.
The specificity of a probe can be diminished by cross-hybridization, also called non-specific hybridization, a phenomenon in which sequences that are not strictly complementary according to the Watson Crick rules bind to each other.
Probe set design was carried out to maximize specificity and minimize cross-hybridization.
The technique showed a significant specificity since no cross-hybridization to M. intracellulare (MIC), M. fortuitum (MFT), M. avium (MAV), M. kansasii (MKS), and M. gordonae (MGD) genomic DNAs was observed.
The use of artificially synthesized oligodeoxyribonucleotides only allows us to quantify the absolute signal intensity without the effect of cross-hybridization and then to evaluate the specificity for single nucleotide mismatches even when the applied target concentration is low.
Out of a total of 247,965 perfect match probes, 95,008 were designated "transcript-specific", i.e. showing complete sequence alignment, no cross-hybridization, and transcript-, nonlynly gene-specificity.
To avoid cross-hybridization, all other alignments are tested using the specificity filtering test.
