Nucleic
Acids Research
Vol. 34, No. 9, 2006; Pages: 1–12
Tests
of rRNA hybridization to microarrays suggest that hybridization
characteristics of oligonucleotide probes for species discrimination
cannot be predicted
Alex Pozhitkov1,3, Peter
A. Noble1, Tomislav Domazet-Loso2, Arne
W. Nolte3, Rainer Sonnenberg3, Peer
Staehler4, Markus Beier4 and Diethard
Tautz3,*
Institute for Genetics, Cologne, D-50674, Germany.
Abstract
Hybridization of rRNAs to microarrays is
a promising approach for prokaryotic and eukaryotic species
identification. Typically, the amount of bound target ismeasuredbyfluorescent
intensityandit isassumed that the signal intensity is directly
related to the target concentration. Using thirteen different
eukaryotic LSU rRNA target sequences and 7693 short perfect
match oligonucleotide probes, we have assessed current approaches
for predicting signal intensities by comparing Gibbs free
energy (Go) calculations
to experimental results. Our evaluation revealed a poor statistical
relationship between predicted and actual intensities. Although
signal intensities for a given target varied up to 70-fold,
none of the predictors were able to fully explain this variation.
Also, no combination of different free energy terms, as assessed
by principal component and neural network analyses, provided
a reliable predictor of hybridization efficiency. We also
examined the effects of single-base pair mismatch (MM) (all
possible types and positions) on signal intensities of duplexes.
We found that the MM effects differ from those that were predicted
from solution-based hybridizations. These results recommend
against the application of probe design software tools that
use thermodynamic parameters to assess probe quality for species
identification. Our results imply that the thermodynamic properties
of oligonucleotide hybridization are by far not yet understood.
Environmental Information System (ENVIS) provides national and international
environmental information in the disciplines of pollution control,microorganisms,envis,
toxic chemicals, central and offshore ecology, environmentally sound and appropriate
technology, bio-degradation of wastes and environment management.
microorganims,envis,environment information system centre, microorganisms
and environment management, environment management, microorganisms, microbes
Environment Information System Centre - microorganims,envis,environment
management, microorganisms, microbes, pollution control, toxic chemicals,
central and offshore ecology, environmentally sound and appropriate technology,
bio-degradation of wastes.
Go) calculations
to experimental results. Our evaluation revealed a poor statistical
relationship between predicted and actual intensities. Although
signal intensities for a given target varied up to 70-fold,
none of the predictors were able to fully explain this variation.
Also, no combination of different free energy terms, as assessed
by principal component and neural network analyses, provided
a reliable predictor of hybridization efficiency. We also
examined the effects of single-base pair mismatch (MM) (all
possible types and positions) on signal intensities of duplexes.
We found that the MM effects differ from those that were predicted
from solution-based hybridizations. These results recommend
against the application of probe design software tools that
use thermodynamic parameters to assess probe quality for species
identification. Our results imply that the thermodynamic properties
of oligonucleotide hybridization are by far not yet understood.