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Secondary structure in the target as a confounding factor in synthetic oligomer microarray design
Vladyslava G Ratushna1, Jennifer W Weller, Cynthia J Gibas
1Department of Biology, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, USA. vratushn@vt.edu <vratushn@vt.edu>
BMC Genomics
|March 10, 2005
Summary
Target secondary structure significantly impacts oligonucleotide probe hybridization in microarrays. Stable structures in target molecules hinder probe binding, affecting results and necessitating design adjustments.
Area of Science:
- Molecular Biology
- Bioinformatics
- Genomics
Background:
- Oligonucleotide probe design typically overlooks target secondary structure.
- Existing software assumes probe secondary structure screening is sufficient.
- Target molecule properties can significantly influence hybridization kinetics and extent in microarrays.
Purpose of the Study:
- To predict and analyze secondary structure formation in a genome-wide set of transcripts.
- To assess the impact of target secondary structure on oligonucleotide probe hybridization.
- To evaluate current probe design software's limitations regarding target structure.
Main Methods:
- Genome-wide transcript secondary structure prediction using thermodynamic analysis.
- Modeling hybridization conditions (high temperature, salt concentration).
- Analysis of probe binding site accessibility within target secondary structures.
Main Results:
- Significant portions of target molecules remain inaccessible due to stable intramolecular secondary structures, even at high temperatures (e.g., 28% of cDNA at 65°C).
- Probe binding sites show substantial double-stranded regions within target structures (e.g., 21% of nucleotides in 70mer probe sites).
- RNA targets exhibit more stable and extensive secondary structures than cDNA; target shearing reduces but does not eliminate secondary structure.
Conclusions:
- Target secondary structure is prevalent and persists even at high temperatures.
- Stable target structures can impede hybridization, impacting microarray data interpretation.
- Incorporating target secondary structure into oligonucleotide design is crucial for optimizing probe performance.