Kotler L, Sobolev I, Ulanovsky L
Department of Structural Biology, Weizmann Institute, Rehovot, Israel.
Biotechniques. 1994 Sep;17(3):554-9.
Here we describe our progress in the development of the technology of DNA sequencing by primer walking based on the modular primer approach, which eliminates the primer synthesis bottleneck. Modular primers are assembled from 5-mers, 6-mers, or 7-mers selected from a presynthesized library of as few as 1000 oligonucleotides. This technology is expected to reduce the time per walk by a factor of 20 to 50, and the cost of DNA sequencing by a factor of 5 to 15. Both time and expense will be saved not only on the primer synthesis per se but, more importantly, as a result of the closed-end automation of the complete cycle of walking sequencing, where no human intervention will be required between the walks. Such closed-end automation has until now been precluded due to the need to synthesize a new primer for each walk. As a part of the closed cycle automation development, this report deals with modular primers used for sequencing with fluorescent dye terminators. The success rate and the quality of automated sequencing with modular primers are found similar to those with the conventional 17-20-base-long primers. One of the advantages of the described technique is simple purification without any proteins that need to be removed.
在此，我们描述了基于模块化引物方法的引物步移DNA测序技术的进展，该方法消除了引物合成瓶颈。模块化引物由从仅1000种寡核苷酸的预合成文库中选择的5聚体、6聚体或7聚体组装而成。预计该技术将使每次步移的时间减少20至50倍，并使DNA测序成本降低5至15倍。不仅在引物合成本身，而且更重要的是，由于步移测序完整周期的封闭式自动化，时间和费用都将节省下来，在步移之间无需人工干预。由于每次步移都需要合成新引物，迄今为止这种封闭式自动化一直无法实现。作为封闭式循环自动化开发的一部分，本报告涉及用于荧光染料终止子测序的模块化引物。发现模块化引物自动测序的成功率和质量与传统的17 - 20个碱基长的引物相似。所述技术的优点之一是无需去除任何蛋白质即可进行简单纯化。
