Primer extension analysis of RNA 5' ends

Primer extension method from Jim Brown's hypercard stack.

The sites of transcription initiation or RNA processing are generally determined by S1 protection or primer extension (PE) analysis. Although S1 protection must be used for 3'-end analysis, 5'-end analysis is usually more easily & precisely done by primer extension analysis. In this method, a short antisense 5' end-labeled DNA primer (usually a synthetic oligonucleotide, but sometimes a small restriction fragment) is hybridized to RNA, usually total cellular RNA, then DNA is synthesized from this primer using reverse transcriptase. RTase will copy the RNA from the site of primer annealing to the 5'-end of the RNA molecule. The reactions are then analyzed by electrophoresis in sequencing gels in lanes adjacent to Sanger sequencing reactions of DNA containing the gene of question, using the same primer as that used in the PE analysis. The transcription initiation site (usually) can then easily be identified as the band in the sequencing reaction directly parallel to the run-off reverse transcript. Multiple transcription initiation sites will appear as multiple bands in the PE lane. RNA processing sites (i.e. in the case of stable RNAs) will also appear in PE analyses, and must be distinguished from transcription initiation sites by other means (usually by examination of the DNA sequence for promoter elements).

Last update 08-Apr-2008, Rating n/a of 1 votes.