Probing the SELEX process with next-generation sequencing.

BACKGROUND: SELEX is an iterative process in which highly diverse synthetic nucleic acid libraries are selected over many rounds to finally identify aptamers with desired properties. However, little is understood as how binders are enriched during the selection course. Next-generation sequencing offers the opportunity to open the black box and observe a large part of the population dynamics during the selection process. METHODOLOGY: We have performed a semi-automated SELEX procedure on the model target streptavidin starting with a synthetic DNA oligonucleotide library and compared results obtained by the conventional analysis via cloning and Sanger sequencing with next-generation sequencing. In order to follow the population dynamics during the selection, pools from all selection rounds were barcoded and sequenced in parallel. CONCLUSIONS: High affinity aptamers can be readily identified simply by copy number enrichment in the first selection rounds. Based on our results, we suggest a new selection scheme that avoids a high number of iterative selection rounds while reducing time, PCR bias, and artifacts.

A streamlined protocol for emulsion polymerase chain reaction and subsequent purification.

Compartmentalization of polymerase chain reaction (PCR) reduces artifacts, especially when complex libraries are amplified. It allows clonal amplification of templates from complex mixtures in a bias-free manner. Here we describe a rapid, straightforward, and easy protocol for PCR in a water-in-oil emulsion (ePCR) including sample recovery by DNA purification. Furthermore, no special laboratory equipment is needed and inexpensive components are used. Therefore, our flexible protocol allows ePCR to be readily implemented in daily routine experiments for a broad range of applications.