Comparison of different amines for the one-lane sequence determination of 5′-end-labeled oligodeoxyribonucleotides by chemical cleavage

Abstract

Hot aqueous solutions of a wide variety of aliphatic amines bring about efficient cleavage of 5′- 32P-labeled oligodeoxyribonucleotides. Electrophoretic resolution of the product mixtures produces a radioactivity pattern that can be analyzed in terms of relative band intensities and band separations to deduce the base sequence of the original oligonucleotide. Amines differ in their overall reactivity with respect to the oligonucleotide as well as in their preference for reaction with the various heterocyclic bases. Guanine sites, in particular, vary markedly in their cleavage susceptibility when reacted with the different amines, being more vulnerable to scission when reacted with less basic amines. Guanine cleavage propensity can also be affected markedly by the ionic strength of the aminolysis solution. Both effects are interpreted in terms of variable extents of deprotonation of the guanine sites in the basic medium. Some of the amines produce fragment patterns that are marred by the presence of minor extraneous bands; these probably are due to an incomplete extent of the second β-elimination involved in backbone cleavage. The methodology is applicable to confirmatory sequencing of synthetic oligonucleotides and can also be used to prepare standard ladders for use in footprinting experiments or chemical reactivity studies.