Poisoned Primer Extension

Poisoned primer extension is primarily used to distinguish between RNAs that are nearly identical in sequence but cannot be distinguished by standard primer extension because they are the same size (e.g., edited vs. nonedited transcripts). It is conceptually identical to the standard primer extension reaction but involves the use of a chain-terminating dideoxynucleotide (the "poison") in the presence of the other three nucleotides. A radioactively labeled primer that hybridizes a short-distance downstream from the "changed" region of interest is extended by reverse transcription into this region of sequence variation. The reactions contain three of the four substrates for extension (e.g., dATP, dGTP, and dTTP) and a chain-terminating dideoxynucleotide (e.g., ddCTP). The extension reaction stops when reverse transcriptase adds a chain-terminating dideoxynucleotide to the template (e.g., it will add ddCTP when it encounters a G in the template sequence). RNAs that differ in sequence at that position will yield different-sized extension products that can be resolved on a denaturing gel.