reMelting curve analysis as a tool for enrichment monitoring in the SELEX process

Abstract

Current aptamer selection procedures enable limited control and transparency on how the DNA selection pool is evolving. Affinity tests and binding analyses are not always informative. Here we show that real-time PCR provides a valuable tool for the follow-up of aptamer selection. Limited time, work and amount of amplified ssDNA make this an interesting instrument to set-up a SELEX design and monitor the enrichment of oligonucleotides. reMelting Curve Analysis (rMCA) after reannealing under stringent conditions provides information about enrichment, compared to a random library. Monitoring the SELEX process and optimising conditions by means of the proposed methods can increase the selection efficiency in a controlled way. rMCA is applied in enrichment simulations and three different selection procedures. Our results imply that rMCA can be used for different SELEX designs and different targets. SELEX pool diversity analysis by rMCA has been proven to be a useful, reproducible tool to detect and evaluate enrichment of specific binding aptamers while the selection procedure is being performed.