Copyright (c) 2009 All rights reserved. http://works.bepress.com/robert_henry Recent documents in Robert J Henry en-us Sun, 15 Nov 2009 15:22:09 PST 3600 http://works.bepress.com/robert_henry/788 http://works.bepress.com/robert_henry/788 Sun, 08 Nov 2009 22:19:23 PST L Slade Lee http://works.bepress.com/robert_henry/787 http://works.bepress.com/robert_henry/787 Sun, 08 Nov 2009 22:19:21 PST 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. Michael J. Cross http://works.bepress.com/robert_henry/786 http://works.bepress.com/robert_henry/786 Sun, 08 Nov 2009 22:19:18 PST L Slade Lee http://works.bepress.com/robert_henry/785 http://works.bepress.com/robert_henry/785 Sun, 08 Nov 2009 20:55:56 PST Susan A. Gillies http://works.bepress.com/robert_henry/784 http://works.bepress.com/robert_henry/784 Sun, 08 Nov 2009 20:55:53 PST Susan Gillies http://works.bepress.com/robert_henry/783 http://works.bepress.com/robert_henry/783 Sun, 08 Nov 2009 16:38:03 PST Peter C. Bundock http://works.bepress.com/robert_henry/782 http://works.bepress.com/robert_henry/782 Tue, 03 Nov 2009 19:21:50 PST A McLauchlan http://works.bepress.com/robert_henry/781 http://works.bepress.com/robert_henry/781 Tue, 03 Nov 2009 19:21:47 PST L Slade Lee http://works.bepress.com/robert_henry/780 http://works.bepress.com/robert_henry/780 Tue, 03 Nov 2009 19:21:45 PST Mervyn Shepherd http://works.bepress.com/robert_henry/779 http://works.bepress.com/robert_henry/779 Tue, 03 Nov 2009 19:21:43 PST Robert J. Henry