Lashkari D A, Hunicke-Smith S P, Norgren R M, Davis R W, Brennan T
Department of Genetics, Stanford University School of Medicine, CA 94305, USA.
Proc Natl Acad Sci U S A. 1995 Aug 15;92(17):7912-5. doi: 10.1073/pnas.92.17.7912.
An automated oligonucleotide synthesizer has been developed that can simultaneously and rapidly synthesize up to 96 different oligonucleotides in a 96-well microtiter format using phosphoramidite synthesis chemistry. A modified 96-well plate is positioned under reagent valve banks, and appropriate reagents are delivered into individual wells containing the growing oligonucleotide chain, which is bound to a solid support. Each well has a filter bottom that enables the removal of spent reagents while retaining the solid support matrix. A seal design is employed to control synthesis environment and the entire instrument is automated via computer control. Synthesis cycle times for 96 couplings are < 11 min, allowing a plate of 96 20-mers to be synthesized in < 5 hr. Oligonucleotide synthesis quality is comparable to commercial machines, with average coupling efficiencies routinely > 98% across the entire 96-well plate. No significant well-to-well variations in synthesis quality have been observed in > 6000 oligonucleotides synthesized to date. The reduced reagent usage and increased capacity allow the overall synthesis cost to drop by at least a factor of 10. With the development of this instrument, it is now practical and cost-effective to synthesize thousands to tens of thousands of oligonucleotides.
已开发出一种自动化寡核苷酸合成仪，它能利用亚磷酰胺合成化学，以96孔微量滴定板的形式同时快速合成多达96种不同的寡核苷酸。一块经过改良的96孔板放置在试剂阀组下方，合适的试剂被输送到含有与固相支持物相连的正在生长的寡核苷酸链的各个孔中。每个孔都有一个滤膜底部，能在保留固相支持物基质的同时去除用过的试剂。采用了一种密封设计来控制合成环境，并且整个仪器通过计算机控制实现自动化。96次偶联的合成循环时间小于11分钟，使得一板96条20聚体寡核苷酸能在不到5小时内合成出来。寡核苷酸的合成质量与商用机器相当，在整个96孔板中，平均偶联效率通常大于98%。在迄今为止合成的6000多种寡核苷酸中，未观察到合成质量在孔与孔之间有显著差异。试剂用量的减少和产量的增加使总体合成成本至少降低了10倍。随着该仪器的开发，现在合成数千到数万个寡核苷酸既切实可行又具有成本效益。
