Liu Wen-Tso, Guo Huiling, Wu Jer-Horng
Division of Environmental Science and Engineering, National University of Singapore, Block E2, no. 04-07, 1 Engineering Drive 2, Singapore 117576.
Appl Environ Microbiol. 2007 Jan;73(1):73-82. doi: 10.1128/AEM.01468-06. Epub 2006 Oct 27.
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 ( approximately 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.
使用一组针对大肠杆菌16S rRNA基因的166个寡核苷酸探针，研究了靶标大小对微阵列杂交效率和特异性的影响。靶标包括未片段化的天然rRNA、片段化的rRNA（约20至100 bp）、PCR扩增子（93至1480 bp）以及三种合成单链DNA寡核苷酸（45至56 bp）。与靶标杂交的探针的荧光强度分为I类（相对于对照探针为81%至100%）、II类（61%至80%）、III类（41%至60%）、IV类（21%至40%）、V类（6%至20%）和VI类（0%至5%）。对于那些赋予I至IV类强度的探针，定义为具有良好的杂交效率；V类和VI类的探针分别被视为弱信号和假阴性信号。使用未片段化的天然rRNA时，13.9%的探针荧光强度在I至IV类，而大多数（57.8%）表现出假阴性信号。对于1480 bp的PCR扩增子也观察到类似趋势（6.6%的探针在I至IV类）。相比之下，片段化rRNA杂交后，I至IV类探针的百分比升至83.1%。同样，当DNA靶标大小从1480 bp减小到45 bp时，该百分比增加了约14倍。总体而言，片段化rRNA（20至100 bp）、短PCR扩增子（<150 bp）和合成靶标（45至56 bp）可提高微阵列杂交效率和特异性。这种认识对于DNA微阵列技术在微生物群落研究中的应用很重要。
