Again, we can pre-design a whole set of such probe sequences optimized for similar hybridization behavior with minimal cross-hybridization
, bothersome secondary structures, or other problems.
The sequence of mature miRNA is generally very short, and there is interference of cross-hybridization
of sequences between homologous miRNAs, so the new challenge is put forward in detection sensitivity and specificity, etc.
Given the presence of sparse cross-hybridization
artifacts in individual microarray probes (Figure 1, panel B), we further tested the samples using a previously reported CHIKV PCR assay (7), which generated results 100% concordant with those of TMA (Figure 1, panel C).
This database must be of high quality in order to obtain the right design and to avoid wrong cross-hybridization
results caused by poor sequences quality and erroneous affiliation in public environmental databases.
The array-platform setup uses unique and distinct ZIP-code probes that show no cross-hybridization
to any known organisms.
Characteristics examined include adequacy and consistency of signal strength (brightness), lack of background and/or cross-hybridization
signals, and presence of appropriate control (internal or external) signals.
One can divide the pattern into 2 regions: cross-hybridization
and normalization (Fig.
The best probes were then selected on the basis of a combination of sequence complexity, base composition, predicted thermodynamic properties, predicted cross-hybridization
, and position within the transcript (Hughes et al.
In the field, the actual cross-hybridization
rate is a function of distance, with percent out-crossing diminishing the farther the recipient is from the pollen source.
These particular mismatches produced no cross-hybridization
. Conversely, the opposite situation (mismatched hybrid P250R probe stabilizer/ P253R template) contains 3 mismatches (2 within the stabilizer and 1 within the probe), none of which involve the critical base-stacking interaction (Fig.
Prototype regions of 1 influenza subtype exhibited no interference from other subtypes (Figure 1A-C), and prototype regions of other pathogens on RPM version 1 showed no cross-hybridization
with any influenza virus segments (Figure 1D).
In addition, there were problems with cross-hybridization
between loci of a given class.