Determination of Sequence Preferences of DNA Binding Proteins Using Pooled Solid-Phase Sequencing of Low Degeneracy Oligonucleotide Mixtures

Mutational analysis via synthetic oligonucleotides, combined with a number of biochemical approaches, has provided a wealth of information on the nature of protein-DNA interactions. Although in vitro mutational studies of the DNA target site were based initially on individual oligonucleotides, recently their power has been greatly enhanced by procedures involving degenerate oligonucleotide mixtures, in which all possible sequence combinations can be represented and from which particular sequences can be selected by protein binding. Strategies based on this approach, however, typically are quite laborious: They involve cloning of the oligonucleotides after the selection step and sequencing of a great number of individual clones to yield statistically significant information.