A new method for sequence analysis of oligodeoxyribonucleotides

Abstract

The structure of an oligodeoxyribonucleotide may be determined by a simple two-dimensional separation on a polyethyleneimine-cellulose thin layer sheet. Chromatography in the first dimension fractionates by chain length a nested set of fragments that are generated by subjecting the oligomer to partial spleen phosphodiesterase degradation and then labelling their non-common ends with 32P using polynucleotide kinase. A subsequent in situ treatment with nuclease Bal 31 produces labelled mononucleotides, and these are identified by chromatography in the second dimension. Since the method does not identify the 3' terminal nucleotide, a convenient procedure involving 3' end labelling followed by enzymatic digestion to monomers has been developed for this purpose. This approach to sequence analysis also has the advantage of permitting assignment of the identity and location of any modified or unusual bases within the oligonucleotide.