Using universal degenerate primers for restriction digestion mapping by PCR

In this study, a polymerase chain reaction (PCR) is developed to determine the restriction map without using restriction endonucleases. A 937 bp fragment of pUC19 which contained one cut site for EcoRI and two recognition sites for PvuII was used as a model. The PCR was carried out using designed degenerate primers and the products were analyzed on 1.5% agarose gel. The number of cut sites, length of fragments and the arrangement of the fragments from 3′ or 5′ end of desired sequence were determined.