An increasing number of proteins are being identified that regulate ge
ne expression by binding specific nucleic acids in vivo. A method term
ed genomic SELEX facilitates the rapid identification of networks of p
rotein-nucleic acid interactions by identifying within the genomic seq
uences of an organism the highest affinity sites for any protein of th
e organism. As with its progenitor, SELEX of random-sequence nucleic a
cids, genomic SELEX involves iterative binding, partitioning, and ampl
ification of nucleic acids. The two methods differ in that the variabl
e region of the nucleic acid library for genomic SELEX is derived from
the genome of an organism. We have used a quick and simple method to
construct Escherichia coil, Saccharomyces cerevisiae, and human genomi
c DNA PCR libraries that can be transcribed with T7 RNA polymerase. We
present evidence that the libraries contain overlapping inserts start
ing at most of the positions within the genome, making these libraries
suitable for genomic SELEX.