We describe an approach to high-throughput parallel DNA synthesis in which a multiwell format is used. The reactions are carried out in open wells using an argon ambient atmosphere to prevent reagent contamination. The controlled-pore glass beads which form the substrate for synthesis are held in individual wells with high-density polyethylene filter bottoms through which reagents are drawn into a vacuum manifold. The synthesis is carried out using direct reagent dispensing into the individual reaction wells. A computer controls the sequence in which reagents are dispensed and the timing of the periodic vacuum pulses required to synthesize the desired sequence. Experiments to date have demonstrated the viability of the approach for a variety of test sequences. Results obtained with HPLC analysis demonstrate coupling efficiencies as high as 99.5% under optimized conditions. Use of the oligomers for DNA sequencing templates and as PCR primers has been demonstrated in production applications. The current instrument design consists of a series of discrete reaction chambers in a 12 channel module which can be multiplexed in a 12 x n format where n can be 1-8, i.e. 96 wells. A projected time interval for 12 parallel syntheses is 2.5 h, with 96 syntheses in 3.5 h. Because of the reduced volume of reagents required in the open well format, significant cost savings are projected.
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机译:我们描述了一种使用多孔格式的高通量并行DNA合成方法。反应在开放的井中使用氩气环境进行,以防止试剂污染。形成用于合成的底物的受控孔玻璃珠被固定在具有高密度聚乙烯过滤器底部的各个孔中,通过这些底部将试剂抽入真空歧管中。使用直接分配到各个反应孔中的试剂进行合成。计算机控制着分配试剂的顺序以及合成所需序列所需的周期性真空脉冲的时间。迄今为止的实验已经证明了该方法在各种测试序列中的可行性。 HPLC分析获得的结果表明,在优化条件下的偶联效率高达99.5%。在生产应用中已证明将寡聚体用于DNA测序模板和作为PCR引物。当前的仪器设计由一系列12通道模块中的离散反应室组成,这些反应室可以12 x n格式多路复用,其中n可以是1-8,即96孔。 12个并行合成的预计时间间隔为2.5小时,其中96个合成在3.5小时内。由于在敞井式中所需的试剂量减少了,因此预计可以节省大量成本。
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