DNA sequencing: modular primers for automated walking

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.