ADAPTER-BASED URACIL DNA GLYCOSYLASE CLONING SIMPLIFIES SHOTGUN LIBRARY CONSTRUCTION FOR LARGE-SCALE SEQUENCING

An improved strategy for the preparation of libraries for the random s equencing of DNA is reported. The protocol is a modification of a prev ious adaptor-based strategy, and utilizes long (11 base) overhangs, wh ich eliminates the unreliable step of vector-insert ligation. The rand om inserts are prepared by adaptor ligation, while the M13 vector is p repared as described for uracil DNA glycosylase (UDG) cloning of polym erase chain reaction (PCR) products, using PCR with uracil-containing primers, followed by UDG treatment to produce overhangs. This method h as been found to reliably yield large numbers of clones. There is no b ackground due to religation of the vector, and all clones contain inse rts. In addition, the method is simple and suitable for export to othe r investigators. Libraries were constructed from cosmids containing hu man DNA and from human cDNAs in order to characterize a strategy for s hotgun sequencing of multiple shorter fragments. (C) 1994 Academic Pre ss, Inc.