Generation of High-Sensitivity Antisense cDNA Probes by Asymmetric PCR

Abstract

Monitoring changing levels of mRNA by hybridization analysis relies on the use of labeled probes. The development of antisense single-stranded cDNA probe methodologies (unidirectional polymerase chain reaction [PCR] amplification or asymmetric PCR amplification) allows the production of probes of high sensitivity and low background with excellent linearity of detection. Because the methods are PCR-based, templates do not need extensive modification: selection of oligonucleotide sequences determines the target amplified. Thus such methods are flexible, being successfully achieved on a variety of templates including recombinant plasmids or dsDNA from reverse-transcription PCR (RT-PCR). In addition these probes are relatively easily stripped from hybridization membranes, so allowing repeated probing in Northern analysis.