In Vitro Selection of Protein-Binding DNA Aptamers as Ligands for Biosensing Applications

Abstract

Aptamers are single-stranded functional nucleic acids that possess cognate ligand recognition capability. These functional nucleic acids have been used for biosensing of a variety of ligands. Aptamers are isolated by "in vitro selection" or SELEX from random-sequence nucleic acid pools. For example, DNA aptamers that recognize a protein can be generated by applying a DNA library to an affinity column containing the protein target and retrieving the bound sequences after wash. These sequences are amplified and used for a new round of binding and amplification. The identity of enriched sequences are subsequently revealed by cloning and sequencing. The binding of individual aptamers to the protein can be confirmed by techniques such as gel mobility shift. This chapter will provide a detailed protocol for isolating protein-binding DNA aptamers.