Xiao Pengfeng, Bu Ying, Zhang Xiaodang, Wu Haiping, Zhou Guohua, Lu Zuhong
Huadong Research Institute for Medicine and Biotechnics, Nanjing 210002, P R. China.
J Nanosci Nanotechnol. 2005 Aug;5(8):1211-5. doi: 10.1166/jnn.2005.205.
In this paper, a simple, reliable and flexible method for fabricating oligonucleotide arrays integrating in in-situ synthesis with a spotting technique is described. In this approach, different oligonucleotide sequences were synthesized on coded modification glass slides using combinatorial chemistry and a mature phosphoramidite chemistry protocol. The slides were then sliced into smaller pieces. Finally, an oligonucleotide array was fabricated by arbitrarily assembling the different coded pieces onto another solid support. A 5 x 5 array including four different sequences of the P16 gene and a control (blank) was successfully assembled. The results indicated that the hybridization fluorescence intensities from the same sequences located at different places on the array were homogeneous and uniform. Background fluorescence was much lower. The fluorescence intensity ratio of a matched sequence to a one-base mismatched sequence, a two-base mismatched sequence and a three-base mismatched sequence was 0.499, 0.236 and 0.04, respectively. The results for the same sequence at different spots in the chip were reproducible with the relative standard deviation ranging from 6.64% to 10.2% (n = 5). This method has the advantages of high probe-density of in-situ synthesis, and off-chip flexibility. Moreover, it does not need any immobilization process to bond oligonucleotides on the substrate.
本文描述了一种将原位合成与点样技术相结合来制备寡核苷酸阵列的简单、可靠且灵活的方法。在这种方法中，使用组合化学和成熟的亚磷酰胺化学方案在编码修饰的载玻片上合成不同的寡核苷酸序列。然后将载玻片切成较小的片。最后，通过将不同的编码片任意组装到另一个固体支持物上制备寡核苷酸阵列。成功组装了一个5×5阵列，包括P16基因的四个不同序列和一个对照（空白）。结果表明，位于阵列不同位置的相同序列的杂交荧光强度是均匀一致的。背景荧光低得多。匹配序列与单碱基错配序列、双碱基错配序列和三碱基错配序列的荧光强度比分别为0.499、0.236和0.04。芯片上不同点相同序列的结果具有可重复性，相对标准偏差为6.64%至10.2%（n = 5）。该方法具有原位合成探针密度高和芯片外灵活性的优点。此外，它不需要任何固定化过程来将寡核苷酸连接到基质上。
