Work

Minimal Primer and Primer-Free SELEX Protocols for Selection of Aptamers from Random DNA Libraries

Year: 2008

Type: article

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.

Source: BioTechniques

DOI: 10.2144/000112689

Authors: Weihua Pan, Ping Xin, Gary A. Clawson

Institutions: Pennsylvania State University, Penn State Milton S. Hershey Medical Center

Language: English

Topic: Advanced biosensing and bioanalysis techniques

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: gold

Estimated APC paid: $1,415

Funders: National Institutes of Health, U.S. Food and Drug Administration, National Cancer Institute