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 (less than or equal to 1000 bp) starti ng from a large cosmid (35,000 bp) containing exogenous DNA. The proce ss is based on restriction enzymatic digestion followed by HPLC separa tion and fractions collection. All DNA fragments are separated in a si ngle run, detected ''on-line'' by UV absorption, and straightforward c ollected with very high recovery. Small fragments can be directly subj ected to the sequence procedure, whereas those larger than 1000 bp are redigested with a second enzyme, the fractionated subfragments are se parated, ligated to plasmid vector, and sequenced. A human genomic cos mid of 35,000 bp (26H7) has been chosen as a model.