Zhang Lei, Hurek Thomas, Reinhold-Hurek Barbara
Laboratory of General Microbiology, Center of Applied Gene Sensoric (CAG), University of Bremen, PO Box 33 04 40, D-28334 Bremen, Germany.
Nucleic Acids Res. 2005 Oct 27;33(19):e166. doi: 10.1093/nar/gni156.
A key issue in applications of short oligonucleotide-based microarrays is how to design specific probes with high sensitivity. Some details of the factors affecting microarray hybridization remain unclear, hampering a reliable quantification of target nucleic acids. We have evaluated the effect of the position of the fluorescent label [position of label (POL)] relative to the probe-target duplex on the signal output of oligonucleotide microarrays. End-labelled single-stranded DNA targets of different lengths were used for hybridization with perfect-match oligonucleotide probe sets targeting different positions of the same molecule. Hybridization results illustrated that probes targeting the labelled terminus of the target showed significantly higher signals than probes targeting other regions. This effect was independent of the target gene, the fluorophore and the slide surface chemistry. Comparison of microarray signal patterns of fluorescently end-labelled, fluorescently internally random-labelled and radioactively end-labelled target-DNAs with the same set of oligonucleotide probes identified POL as a critical factor affecting signal intensity rather than binding efficiency. Our observations define a novel determinant for large differences of signal intensities. Application of the POL effect may contribute to better probe design and data interpretation in microarray applications.
基于短寡核苷酸的微阵列应用中的一个关键问题是如何设计具有高灵敏度的特异性探针。影响微阵列杂交的一些因素细节仍不清楚，这妨碍了对靶核酸进行可靠的定量分析。我们评估了荧光标记位置[标记位置（POL）]相对于探针-靶标双链体对寡核苷酸微阵列信号输出的影响。使用不同长度的末端标记单链DNA靶标与靶向同一分子不同位置的完全匹配寡核苷酸探针组进行杂交。杂交结果表明，靶向靶标标记末端的探针显示出比靶向其他区域的探针明显更高的信号。这种效应与靶基因、荧光团和玻片表面化学性质无关。用同一组寡核苷酸探针比较荧光末端标记、荧光内部随机标记和放射性末端标记的靶DNA的微阵列信号模式，确定POL是影响信号强度而非结合效率的关键因素。我们的观察结果确定了信号强度存在巨大差异的一个新决定因素。POL效应的应用可能有助于在微阵列应用中进行更好的探针设计和数据解读。
