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Applied and Environmental Microbiology, January 2007, p. 73-82, Vol. 73, No. 1
0099-2240/07/$08.00+0     doi:10.1128/AEM.01468-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Effects of Target Length on the Hybridization Efficiency and Specificity of rRNA-Based Oligonucleotide Microarrays{triangledown} ,{dagger}

Wen-Tso Liu,* Huiling Guo, and Jer-Horng Wu{ddagger}

Division of Environmental Science and Engineering, National University of Singapore, Singapore 117576

Received 26 June 2006/ Accepted 17 October 2006

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.

* Corresponding author. Mailing address: Division of Environmental Science and Engineering, National University of Singapore, Block E2, no. 04-07, 1 Engineering Drive 2, Singapore 117576. Phone: (65) 65161315. Fax: (65) 67744202. E-mail: eseliuwt{at}nus.edu.sg.

{triangledown} Published ahead of print on 27 October 2006.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

{ddagger} Present address: Sustainable Environment Research Center, National Cheng Kung University, Taiwan.

Applied and Environmental Microbiology, January 2007, p. 73-82, Vol. 73, No. 1
0099-2240/07/$08.00+0     doi:10.1128/AEM.01468-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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