Detection of Mutations Using a Novel Endonuclease

摘要

We discovered a nuclease, CEL I from celery, that has high specificity for DNA mismatch, including base-substitutions, insertions and deletions. It has been used to develop a robust mutation detection assay. The substrate is fluorescently labeled PCR product heteroduplexes in which the two strands are of two different colors. CEL I nicks one strand of some molecules to produce a truncated DNA band visualized by an automated DNA sequencer fluorescence-based Genescan analysis. In other molecules, the enzyme nicks at the mismatch in the other strand, and produces a band of the second color. The sizes of the two DNA bands independently pinpoint the location of the mismatch. We have optimized this assay, and demonstrated its utility in the screening of mutations and polymorphisms. Our test samples include all the coding exons of the BRCAl gene of 10 patients, 500 basepairs of exon 11 of 100 patients, and all the exons of the ARSC gene of 100 individuals. Both of these genes are important to breast cancer. To further economize the assay, we validated a protocol to use a single pair of fluorescent primers in a nested PCR a roach to screen all the exons of the ARSC gene of 100 individuals.