Table of Contents
ISRN Molecular Biology
Volume 2012, Article ID 939083, 9 pages
http://dx.doi.org/10.5402/2012/939083
Research Article

Real-Time PCR-Coupled CE-SELEX for DNA Aptamer Selection

School of Biology, Georgia Institute of Technology, 310 Ferst Drive, Atlanta, GA 30332, USA

Received 3 May 2012; Accepted 17 June 2012

Academic Editors: J. Ciesiolka, G. V. Gursky, H. A. Heus, and S. N. Richter

Copyright © 2012 Patrick Ruff et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Aptamers are short nucleic acid or peptide sequences capable of binding to a target molecule with high specificity and affinity. Also known as “artificial antibodies,” aptamers provide many advantages over antibodies. One of the major hurdles to aptamer isolation is the initial time and effort needed for selection. The systematic evolution of ligands by exponential enrichment (SELEX) is the traditional procedure for generating aptamers, but this process is lengthy and requires a large quantity of target and starting aptamer library. A relatively new procedure for generating aptamers using capillary electrophoresis (CE), known as CE-SELEX, is faster and more efficient than SELEX but requires laser-induced fluorescence (LIF) to detect the aptamer-target complexes. Here, we implemented an alternative system without LIF using real-time- (RT-) PCR to indirectly measure aptamer-target complexes. In three rounds of selection, as opposed to ten or more rounds common in SELEX protocols, a specific aptamer for bovine serum albumin (BSA) was obtained. The specificity of the aptamer to BSA was confirmed by electrophoretic mobility shift assays (EMSAs), an unlabeled competitor assay, and by a supershift assay. The system used here provides a cost effective and a highly efficient means of generating aptamers.