IDENTIFICATION OF MODIFIED BASES AND THEIR LOCATIONS IN DNA (NUCLEOTIDES)
Abstract
I. The locations of modified and common bases in 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 followed by labelling their non-common ends with ('32)P 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. II. A two-dimensional method which can be used in conjunction with a standard sequencing technique to identify the positions of modified bases in polydeoxyribonucleotides is described. In the first dimension, a set of DNA fragments is separated by length, and in the second dimension, the 5' termini are identified. The method consists of several steps: partial digestion of the DNA with nuclease BAL 31, removal of 5' phosphates with calf intestinal phosphatase, radioactive labelling of the 5' termini with polynucleotide kinase, asymmetric cleavage by a restriction endonuclease to generate a short nested set of fragments and a long nested set of fragments, separation of the short fragments by length using polyacrylamide gel electrophoresis, blot-transfer of the fragments to a polyethyleneimine thin layer sheet, in situ digestion to monomers using nuclease Bal 31, and fractionation of the monomers by thin layer chromatography. Autoradiography then shows a pattern of spots corresponding to the nucleotides, including those with modified bases, at each position of the polynucleotide chain. In case the resolution of the spots after the two-dimensional separation does not allow precise location of a particular base, comparison of the autoradiograph with the sequence as determined by a standard technique allows the location to be determined without ambiguity.
Degree
Ph.D.
Subject Area
Molecular biology
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.