MUTATION DETECTION USING A NOVEL PLANT ENDONUCLEASE

We have discovered a useful new reagent for mutation detection, a nove l nuclease CEL I from celery. It is specific for DNA distortions and m ismatches from pH 6 to 9, Incision is on the 3'-side of the mismatch s ite in one of the two DNA strands in a heteroduplex. CEL I-like nuclea ses are found in many plants. We report here that a simple method of e nzyme mutation detection using CEL I can efficiently identify mutation s and polymorphisms. To illustrate the efficacy of this approach, the exons of the BRCA1 gene were amplified by PCR using primers 5'-labeled with fluorescent dyes of two colors, The PCR products were annealed t o form heteroduplexes and subjected to CEL I incision. In GeneScan ana lyses with a PE Applied Biosystems automated DNA sequencer, two indepe ndent incision events, one in each strand, produce truncated fragments of two colors that complement each other to confirm the position of t he mismatch. CEL I can detect 100% of the sequence variants present, i ncluding deletions, insertions and missense alterations, Our results i ndicate that CEL I mutation detection is a highly sensitive method for detecting both polymorphisms and disease-causing mutations in DNA fra gments as long as 1120 bp in length.