Abstract
Writing DNA plays a significant role in the fields of synthetic biology, functional genomics and bioengineering. DNA clones on next-generation sequencing (NGS) platforms have the potential to be a rich and cost-effective source of sequence-verified DNAs as a precursor for DNA writing. However, it is still very challenging to retrieve target clonal DNA from high-density NGS platforms. Here we propose an enabling technology called ‘Sniper Cloning’ that enables the precise mapping of target clone features on NGS platforms and non-contact rapid retrieval of targets for the full utilization of DNA clones. By merging the three cutting-edge technologies of NGS, DNA microarray and our pulse laser retrieval system, Sniper Cloning is a week-long process that produces 5,188 error-free synthetic DNAs in a single run of NGS with a single microarray DNA pool. We believe that this technology has potential as a universal tool for DNA writing in biological sciences.
Highlights
Writing DNA plays a significant role in the fields of synthetic biology, functional genomics and bioengineering
The next-generation sequencing (NGS) platform GS Junior from Roche 454 Life Sciences identifies the content of the complex pool of DNAs from the microarray through in vitro cloning followed by massively parallel pyrosequencing
We developed a ‘diffusion-like local mapping algorithm’ to pinpoint the exact location of the target clone beads on the substrate, and selectively separated the beads containing the desired sequence-verified oligonucleotides for direct utilization (Fig. 1b)
Summary
Writing DNA plays a significant role in the fields of synthetic biology, functional genomics and bioengineering. By merging the three cutting-edge technologies of NGS, DNA microarray and our pulse laser retrieval system, Sniper Cloning is a week-long process that produces 5,188 error-free synthetic DNAs in a single run of NGS with a single microarray DNA pool. We believe that this technology has potential as a universal tool for DNA writing in biological sciences. In spite of the significant throughput enhancement in the synthesis process, selecting the correct nucleotides from the highly mixed and complex pool of error-prone oligonucleotides severely increases the expense and obscures the major benefits of microarray synthesis. Conventional in vivo cloning has little utility in the purification of the complex pool of oligonucleotides because of the insufficient throughput of fully randomized pickand-place colony selection followed by Sanger sequencing
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