title: Minimal primer and primer-free SELEX protocols for selection of aptamers from random DNA libraries.
abstract: Standard systematic evolution of ligands by exponential enrichment (SELEX) protocols require libraries that contain two primers, one on each side of a central random domain, which allow amplification of target-bound sequences via PCR or RT-PCR. However, these primer sequences cause nonspecific binding by their nature (generally adding about 20 nt on each end of the random sequence of about 30-40 nt), and can result in large numbers of false-positive binding sequences and/or interfere with good binding random sequences. Here, we have developed two DNA-based methods that reduce and/or eliminate the primer sequences from the target-binding step, thus reducing or eliminating the interference caused by the primer sequences. In these methods, the starting selection libraries contain a central random sequence that is: (i) flanked by only 2 nt on each side (minimal primer); or (ii) flanked only by either a 2- or 0-nt overhand on the 3' end (primer-free). These methods allow primer regeneration and re-elimination after and before selection, are fast and simple, and don't require any chemical modifications for selection in a variety of conditions. Further, the selection rounds are performed with DNA oligomers, which are generally employed as end product aptamers.
authors: Gittlen Cancer Research Foundation, Hershey Medical Center, Department of Pathology, College of Medicine, Pennsylvania State University, Hershey, PA 17033, USA.
authors: Weihua
authors: W
authors: Pan
authors: Ping
authors: P
authors: Xin
authors: Gary A
authors: GA
authors: Clawson
mesh_terms: D052157:Aptamers, Nucleotide / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics*; D015723:Gene Library; D016133:Polymerase Chain Reaction / Q000379:methods*; D052156:SELEX Aptamer Technique / Q000379:methods*
chemical_list: D052157:Aptamers, Nucleotide; D017931:DNA Primers