S1 analysis of messenger RNA using single-stranded DNA probes

This method takes advantage of the ability of oligonucleotides to be efficiently labeled to a high specific activity at the 5' end through the use of kinase. The oligonucleotide is hybridized to a specific single-stranded template containing the complementary sequence to the oligonucleotide, and this hybrid is extended through the use of the Klenow fragment of E. coli DNA polymerase I. The mixture is cut with a restriction enzyme to give the probe a defined 3' end, and the probe is isolated on an alkaline agarose gel. Before using this protocol it is first helpful to have an M13 clone. If this is unavailable, a double-stranded plasmid clone of the region to be studied may be used, as described in an alternate protocol. Another alternate protocol describes the use of long oligonucleotides as probes for S1 analysis (useful for rapid and easy quantitation of the level of mRNA produced from a characterized promoter). For the mapping of the 5' end of an RNA species, hybridization of the probe to RNA is then carried out. S1 nuclease is added to digest all of the unhybridized portion of the probe. Electrophoresis of the hybrid on a denaturing polyacrylamide gel allows a determination of the length of the remaining DNA fragment. This length equals the distance between the 5' end of the probe to the 5' end of the RNA, defining the transcriptional start site to the nucleotide. By performing the hybridization reaction in vast probe excess, quantitation of the relative amounts of RNA can be estimated between samples.