SHORT-INSERT LIBRARIES AS A METHOD OF PROBLEM-SOLVING IN GENOME SEQUENCING

As the Human Genome Project moves into its sequencing phase, a serious problem has arisen. The same problem has been increasingly vexing in the closing phase of the Caenorhabditis elegans project. The difficult y lies in sequencing efficiently through certain regions in which the templates (DNA substrates for the sequencing process) form complex fol ded secondary structures that are inaccessible to the enzymes. The sol ution, however, is simply to break them up. Specifically, the offendin g fragments are sonicated heavily and recloned, as much smaller fragme nts, into pUC vector. The sequences obtained from the resulting librar y can subsequently be assembled, free from the effects of secondary st ructure, to produce high-quality, complete sequence. Because of the su ccess and simplicity of this procedure, we have begun to use it for th e sequencing of all regions in which standard primer walking has been at all difficult.