We have developed a method to remove abundant sequences from cDNA libraries based on second-order kinetics of reassociation of double-stranded cDNAs. First, the same set of dauer larva-specific cDNAs (unnormalized cDNAs) was cloned into two vectors - lambda
gt11 and lambda ZapII. Second, the cDNA inserts of recombinant lambda ZapII molecules were amplified by PCR and used for preparation of an abundant cDNA pool. The preparation of this pool involved denaturation of the double-stranded cDNA inserts, partial reassociation, and selection of the double-stranded fraction. Single-stranded unnormalized cDNAs were prepared from the lambda
gt11 library and mixed with an excess of the abundant cDNA pool. After denaturation and partial reassociation, the single-stranded fraction was selected. This fraction included single-stranded cDNAs generated from recombinant lambda
gt11 molecules that did not hybridize with abundant cDNAs, as well as nonhybridized single-stranded cDNAs generated from the abundant cDNA pool. Using primers specific for lambda
gt11, the single-stranded cDNAs were amplified by PCR and cloned. We have prepared and characterized two rare cDNA libraries, differing in the degree to which abundant sequences have been depleted. One of them exhibited a 50-100x increase in concentration of
daf-1,
daf-4, and
daf-7 cDNAs along with a 3-fold decrease in concentration of the abundant Hsp90 cDNA (
daf-7 is expressed at low levels in only two cells), relative to the unnormalized cDNA library. The second, more heavily depleted cDNA library exhibited a 3-6x increase in concentration of
daf-1,
daf-4, and
daf-7 cDNAs and at the same time a 35-fold decrease in concentration of Hsp90 cDNA. In this latter cDNA library, the daf sequences were sufficiently abundant not to be enriched as much as in the former cDNA library. We would expect that cDNAs more rare than daf cDNAs should be more highly enriched. We have sequenced 10 random clones of the latter rare cDNA library. Half of these correspond to genes that are not represented by any ESTs in the C. elegans EST database. Such cDNA libraries enriched in rare cDNA sequences can serve as a resource for finding new ESTs and for construction of subtractive cDNA libraries to investigate dauer-specific gene expression.