Improved procedure for the preparation of DNA restriction fragments suitable for sequencing

A rapid and integrated procedure was developed for the preparation of small DNA restriction fragments (< or = 1000 bp) starting from a large cosmid (35,000 bp) containing exogenous DNA. The process is based on restriction enzymatic digestion followed by HPLC separation and fractions collection. All DNA fragments are separated in a single run, detected "on-line" by UV absorption, and straightforward collected with very high recovery. Small fragments can be directly subjected to the sequence procedure, whereas those larger than 1000 bp are redigested with a second enzyme, the fractionated subfragments are separated, ligated to plasmid vector, and sequenced. A human genomic cosmid of 35,000 bp (26H7) has been chosen as a model.