Wei Hairong, Kuan Pei Fen, Tian Shulan, Yang Chuhu, Nie Jeff, Sengupta Srikumar, Ruotti Victor, Jonsdottir Gudrun A, Keles Sunduz, Thomson James A, Stewart Ron
WiCell Research Institute, PO Box 7365, Madison, WI 53707-7365, USA.
Nucleic Acids Res. 2008 May;36(9):2926-38. doi: 10.1093/nar/gkn133. Epub 2008 Apr 1.
Well-defined relationships between oligonucleotide properties and hybridization signal intensities (HSI) can aid chip design, data normalization and true biological knowledge discovery. We clarify these relationships using the data from two microarray experiments containing over three million probes from 48 high-density chips. We find that melting temperature (T(m)) has the most significant effect on HSI while length for the long oligonucleotides studied has very little effect. Analysis of positional effect using a linear model provides evidence that the protruding ends of probes contribute more than tethered ends to HSI, which is further validated by specifically designed match fragment sliding and extension experiments. The impact of sequence similarity (SeqS) on HSI is not significant in comparison with other oligonucleotide properties. Using regression and regression tree analysis, we prioritize these oligonucleotide properties based on their effects on HSI. The implications of our discoveries for the design of unbiased oligonucleotides are discussed. We propose that isothermal probes designed by varying the length is a viable strategy to reduce sequence bias, though imposing selection constraints on other oligonucleotide properties is also essential.
寡核苷酸特性与杂交信号强度（HSI）之间明确的关系有助于芯片设计、数据标准化以及真正生物学知识的发现。我们利用来自两个微阵列实验的数据来阐明这些关系，这两个实验包含来自48个高密度芯片的超过三百万个探针。我们发现，解链温度（T(m)）对HSI的影响最为显著，而在所研究的长寡核苷酸中，长度的影响非常小。使用线性模型对位置效应进行分析表明，探针的突出端对HSI的贡献比连接端更大，这通过专门设计的匹配片段滑动和延伸实验得到了进一步验证。与其他寡核苷酸特性相比，序列相似性（SeqS）对HSI的影响并不显著。通过回归和回归树分析，我们根据这些寡核苷酸特性对HSI的影响对其进行了优先级排序。讨论了我们的发现对无偏寡核苷酸设计的意义。我们提出，通过改变长度设计等温探针是减少序列偏差的可行策略，尽管对其他寡核苷酸特性施加选择限制也至关重要。
