ABSTRACT
Analyte Binding ................................................................. 178 8.2.2 Bioimaging........................................................................................ 179 8.2.3 Afnity Purication Using Aptamers .............................................. 179
8.3 Technical Aspects of SELEX ....................................................................... 180 8.3.1 ssDNA Library and Primers Design ................................................. 180 8.3.2 Targets ............................................................................................... 181 8.3.3 Generation of dsDNA from ssDNA Random Pool ........................... 182 8.3.4 Generation of ssDNA or RNA from dsDNA .................................... 183 8.3.5 Target-Nucleic Acid Interaction ....................................................... 183 8.3.6 Separation ......................................................................................... 184 8.3.7 Amplication .................................................................................... 185 8.3.8 Negative and Positive Selections ...................................................... 185 8.3.9 Rening the Aptamer ....................................................................... 186 8.3.10 Improving Aptamer Afnity by Reselection .................................... 187
8.4 Aptamer Modication................................................................................... 187 8.5 Advances and Variations in SELEX Technology ......................................... 188 8.6 Computational Aids ...................................................................................... 189 8.7 A SELEX Protocol ....................................................................................... 190
8.7.1 Synthesis of dsDNA from ssDNA Library ....................................... 190 8.7.1.1 PCR Reaction (100 μL Total Volume) ............................... 191 8.7.1.2 Taq DNA Polymerase Extension Reaction ........................ 191
Nucleic acid aptamers are short single-stranded oligonucleotides that bind their target molecules avidly and specically. The procedure for in vitro selection of aptamers, called SELEX (systematic evolution of ligands by exponential enrichment), was rst published independently by the Szostak and Gold groups (Figure 8.1).1,2 Over the past 20 years, aptamers have emerged as important biological tools, with high potential as diagnostic and therapeutic reagents as summarized in a recent aptamer handbook.3 Here, we describe the principles and a detailed protocol for SELEX to aid the further development of these promising reagents.
