Optimized quantitative PCR analysis of random DNA aptamer libraries.

ABSTRACT: The quantitative polymerase chain reaction (qPCR) with detection of duplex DNA yield by intercalator fluorescence is a common and essential technique in nucleic acid analysis. We encountered unexpected results when applying standard qPCR methods to the quantitation of random DNA libraries flanked by regions of fixed sequence, a configuration essential for in vitro selection experiments. Here we describe the results of experiments revealing why conventional qPCR methods can fail to allow automated analysis in such cases, and simple solutions to this problem. In particular we show that renaturation of PCR products containing random regions is incomplete in late PCR cycles when extension fails due to reagent depletion. Intercalator fluorescence can then be lost at standard interrogation tempe

SUBMITTER: Pearson K 

PROVIDER: S-EPMC9542921 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature