Construction of a cosmid library of DNA replicated early in the S phase ofnormal human fibroblasts

We constructed a subgenomic cosmid library of DNA replicated early in the S phase of normal human diploid fibroblasts. Cells were synchronized by rele ase from confluence arrest and incubation in the presence of aphidicolin. B romodeoxyuridine (BrdUrd) was added to aphidicolin-containing medium to lab el DNA replicated as cells entered S phase. Nuclear DNA was partially diges ted with Sau 3Al, and hybrid density DNA was separated in CsCl gradients. T he purified early-replicating DNA was cloned into sCos1 cosmid vector. Clon es were transferred individually into ther wells of 96 microtiter plates (9 ,216 potential clones). Vigorous bacterial growth was detected in 8,742 of those wells. High-density colony hybridization filters (1,536 clones/filter ) were prepared from a set of replicas of the original plates. Bacteria rem aining in the wells of replica plates were combined, mixed with freezing me dium, and stored at -80 degrees C. These pooled stocks were analyzed by pol ymerase chain reaction to determine the presence of specific sequences in t he library. Hybridization of high-density filters was used to identify the clones of interest, which were retrieved from the frozen cultures in the 96 -well plates. In testing the library for the presence of 14 known early-rep licating genes, we found sequences at or near 5 of them: APRT, beta-actin, beta-tubulin, c-myc, and HPRT. This library is a valuable resource for the isolation and analysis of certain DNA sequences replicated at the beginning of S phase, including potential origins of bidirectional replication. (C) 2000 Wiley-Liss, Inc.