Please use this identifier to cite or link to this item: https://doi.org/10.1128/AEM.01468-06
Title: Effects of target length on the hybridization efficiency and specificity of rRNA-based oligonucleotide microarrays
Authors: Liu, W.-T. 
Guo, H.
Wu, J.-H. 
Issue Date: Jan-2007
Source: Liu, W.-T., Guo, H., Wu, J.-H. (2007-01). Effects of target length on the hybridization efficiency and specificity of rRNA-based oligonucleotide microarrays. Applied and Environmental Microbiology 73 (1) : 73-82. ScholarBank@NUS Repository. https://doi.org/10.1128/AEM.01468-06
Abstract: The effect of target size on microarray hybridization efficiencies and specificity was investigated using a set of 166 oligonucleotide probes targeting the 16S rRNA gene of Escherichia coli. The targets included unfragmented native rRNA, fragmented rRNA (∼20 to 100 bp), PCR amplicons (93 to 1,480 bp), and three synthetic single-stranded DNA oligonucleotides (45 to 56 bp). Fluorescence intensities of probes hybridized with targets were categorized into classes I (81 to 100% relative to the control probe), II (61 to 80%), III (41 to 60%), IV (21 to 40%), V (6 to 20%), and VI (0 to 5%). Good hybridization efficiency was defined for those probes conferring intensities in classes I to IV; those in classes V and VI were regarded as weak and false-negative signals, respectively. Using unfragmented native rRNA, 13.9% of the probes had fluorescence intensities in classes I to IV, whereas the majority (57.8%) exhibited false-negative signals. Similar trends were observed for the 1,480-bp PCR amplicon (6.6% of the probes were in classes I to IV). In contrast, after hybridization of fragmented rRNA, the percentage of probes in classes I to IV rose to 83.1%. Likewise, when DNA target sizes were reduced from 1,480 bp to 45 bp, this percentage increased approximately 14-fold. Overall, microarray hybridization efficiencies and specificity were improved with fragmented rRNA (20 to 100 bp), short PCR amplicons (< 150 bp), and synthetic targets (45 to 56 bp). Such an understanding is important to the application of DNA microarray technology in microbial community studies. Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Source Title: Applied and Environmental Microbiology
URI: http://scholarbank.nus.edu.sg/handle/10635/67643
ISSN: 00992240
DOI: 10.1128/AEM.01468-06
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