A novel detection strategy for scanning multiple mutations using CEL I

Michael J. Cross, Southern Cross University
L Slade Lee, Southern Cross University
Robert J. Henry, Southern Cross University

Abstract

CEL I endonuclease cleavage of heteroduplex DNA is noted as an efficient technique for scanning homologous sequences for subtle variations. A significant shortcoming of the CEL I technique, however, is the poor signal-to-noise ratio obtained when using a PCR-based, 5’end-labeled, detection platform. Normally, the sensitivity of fluorescent-based electrophoresis is many fold superior to that of ethidium bromide-stained/ UV-visualized agarose electrophoresis. Recent CEL I literature, however, reports that maximum pooling ratios are overall similar, when comparing end-labeled and ethidium bromide-stained detection systems. CEL I is hypothesized to reduce signal strength of end-labeled amplicons, via non-specific exonucleolytic activity, which acts to cleave off 5’ end bases and their linked dyes. Typical CEL I protocols involve only short digestion times in an attempt to limit the loss of true-nicked signal via any undesired exonucleolytic activity. Our research provides strong evidence to support the ‘exonucleolytic cleavage’ hypothesis on CEL I’s activity. We investigate alternative dye-labeling strategies and report a simple, improved technique for CEL I mutation scanning. The final result is an increase in CEL I scanning sensitivity with higher throughputs being enabled via a greater capacity to pool amplified sequences. Our method was developed and tested in rice, across a range of cultivars highly characterized for their SNP content in exon 8 of starch sythase IIa.