Isolating and sequencing the predominant 5'-ends of a specific mRNA in cells. I. Purification by filter hybridization

Procedures are considered for purification of a specific procaryotic RNA by successive hybridizations to DNA immobilized to nitrocellulose with special consideration of problems associated with subsequent end-labeling in the T4 polynucleotide kinase reaction. (1) Inhibitors of the kinase can be associated with the plasmid but were removed by electrophoresis of the DNA fragment through polyacrylamide. (2) Residual soluble acrylamide, contaminating the DNA and preventing its efficient retention to nitrocellulose, could be removed by DE52 chromatography. (3) Short denatured DNA required high salt (0.9 M) to bind to nitrocellulose but reannealed quickly at those salt concentrations unless applied at less than or equal to 0.3 micrograms/ml at 4 degrees C with a flow rate of 1 ml/min. (4) The kinetics of the hybrid reaction were a function of DNA length, concentration, and temperature. (5) Formamide was a more effective denaturing agent to remove hybrid RNA from the filter than either 12 M urea or 8 M guanidine-HCl, but caused significant release of DNA from the nitrocellulose as well as another potent inhibitor of the kinase reaction. The release of DNA and other kinase inhibitors was greatly reduced by eluting in boiling water.