Optimization of polyacrylamide gel electrophoresis conditions used for sequencing mixed oligodeoxyribonucleotides.

Abstract

We investigated two components of the polyacrylamide gel electrophoresis system used for sequencing DNA to improve the system for sequencing synthesized oligodeoxyribonucleotides containing positions of degeneracy. First, we varied the ratio of methylene-bis-acrylamide (MBA) to acrylamide from that commonly used in DNA sequencing gels (1% MBA:19% acrylamide). A moderate increase in the MBA:acrylamide ratio proves optimal when sequencing is used to confirm that a synthesized fragment contains equivalent stoichiometries of the different nucleotides at a given position of degeneracy. Such information is particularly important if the degenerate oligodeoxyribonucleotide preparation is to be employed as a hybridization probe. A further increase in the MBA:acrylamide ratio (3% MBA:19% acrylamide) produces a gel in which the sequence of an oligodeoxyribonucleotide mixture containing several positions of degeneracy can be read most easily. Increasing the MBA acrylamide ratio suppresses the effect of base composition on electrophoretic mobility of a fragment. Second, we investigated the use of a Tris-citrate buffer system in place of the standard Tris-borate system. We found the Tris-citrate system to be significantly more effective in preventing discontinuities in the banding pattern of the smaller fragments. Finally, we show that high MBA gels are also effective in resolving mixtures of oligoribonucleotides such as those produced by T1 ribonuclease digestion of small RNAs.