Adaptor-based uracil DNA glycosylase cloning simplifies shotgun library construction for large-scale sequencing.

An improved strategy for the preparation of libraries for the random sequencing of DNA is reported. The protocol is a modification of a previous adaptor-based strategy, and utilizes long (11 base) overhangs, which eliminates the unreliable step of vector-insert ligation. The random inserts are prepared by adaptor ligation, while the M13 vector is prepared as described for uracil DNA glycosylase (UDG) cloning of polymerase chain reaction (PCR) products, using PCR with uracil-containing primers, followed by UDG treatment to produce overhangs. This method has been found to reliably yield large numbers of clones. There is no background due to religation of the vector, and all clones contain inserts. In addition, the method is simple and suitable for export to other investigators. Libraries were constructed from cosmids containing human DNA and from human cDNAs in order to characterize a strategy for shotgun sequencing of multiple shorter fragments.