CEL I endonuclease cleavage of heteroduplex DNA is noted as an efficient, high-throughput 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-fluorophore-labelled detection platform. Normally, the sensitivity of fluorescent-based PAGE/CE is many fold superior to the UV visualization of intercalated dyes following agarose electrophoresis. Recent literature, however, reports that maximum pooling capacities are overall similar, when comparing sophisticated fluorophore-labelled detection platforms with more basic ethidium-stained techniques. CEL I has been hypothesized to reduce signal strength of end-labelled amplicon fragments via non-specific exonucleolytic activity, which acts to cleave off 5’-end bases and their covalently-linked fluorophores. 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-labelling strategies and report a simple, improved technique for CEL I mutation scanning using an Applied Biosystems 3730 DNA Analyzer. Increased sensitivity is achieved, with higher throughputs being enabled via a greater capacity to pool amplified DNA. The CEL I-scanning protocol is suitable for detecting more than one unique mutant allele per pool. 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.
|Publication status||Published - 2007|
|Event||Plant and Animal Genomes XV Conference - |
Duration: 13 Jan 2007 → …
|Conference||Plant and Animal Genomes XV Conference|
|Period||13/01/07 → …|