Selection of aptamers by systematic evolution of ligands by exponential enrichment: addressing the polymerase chain reaction issue.
Aptamers are DNA oligonucleotides capable of binding different classes of targets with high affinity and selectivity. They are particularly attractive as affinity probes in multiplexed quantitative analysis of proteins. Aptamers are typically selected from large libraries of random DNA sequences in a general approach termed systematic evolution of ligands by exponential enrichment (SELEX). SELEX involves repetitive rounds of two processes: (i) partitioning of aptamers from non-aptamers by an affinity method and (ii) amplification of aptamers by the polymerase chain reaction (PCR). New partitioning methods, which are characterized by exceptionally high efficiency of partitioning, have been recently introduced. For the overall SELEX procedure to be efficient, the high efficiency of new partitioning methods has to be matched by high efficiency of PCR. Here we present the first detailed study of PCR amplification of random DNA libraries used in aptamer selection. With capillary electrophoresis as an analytical tool, we found fundamental differences between PCR amplification of homogeneous DNA templates and that of large libraries of random DNA sequences. Product formation for a homogeneous DNA template proceeds until primers are exhausted. For a random DNA library as a template, product accumulation stops when PCR primers are still in excess of the products. The products then rapidly convert to by-products and virtually disappear after only 5 additional cycles of PCR. The yield of the products decreases with the increasing length of DNA molecules in the library. We also proved that the initial number of DNA molecules in PCR mixture has no effect on the by-products formation. While the increase of the Taq DNA polymerase concentration in PCR mixture selectively increases the yield of PCR products. Our findings suggest that standard procedures of PCR amplification of homogeneous DNA samples cannot be transferred to PCR amplification of random DNA libraries: to ensure efficient SELEX, PCR has to be optimized for the amplification of random DNA libraries.
Musheev
Michael U
MU
Department of Chemistry, York University, Toronto, Ont., Canada M3J 1P3.
Krylov
Sergey N
SN