Copyright (c) 2009 All rights reserved. http://works.bepress.com/slade_lee Recent documents in L Slade Lee en-us Mon, 09 Nov 2009 23:21:53 PST 3600 http://works.bepress.com/slade_lee/166 http://works.bepress.com/slade_lee/166 Sun, 08 Nov 2009 21:54:02 PST L Slade Lee http://works.bepress.com/slade_lee/165 http://works.bepress.com/slade_lee/165 Sun, 08 Nov 2009 21:54:01 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/slade_lee/164 http://works.bepress.com/slade_lee/164 Sun, 08 Nov 2009 21:54:00 PST L Slade Lee http://works.bepress.com/slade_lee/163 http://works.bepress.com/slade_lee/163 Tue, 03 Nov 2009 18:51:26 PST L Slade Lee http://works.bepress.com/slade_lee/162 http://works.bepress.com/slade_lee/162 Tue, 03 Nov 2009 18:51:24 PST L Slade Lee http://works.bepress.com/slade_lee/161 http://works.bepress.com/slade_lee/161 Tue, 03 Nov 2009 18:51:23 PST Maurizio Rossetto http://works.bepress.com/slade_lee/160 http://works.bepress.com/slade_lee/160 Sat, 31 Oct 2009 00:37:41 PDT The Australian Agriculture Research Institute is embarking on a large-scale grape EST analysis project. cDNA libraries are being prepared from a range of tissues, developmental stages and cultivars with the aim of producing a data base of up to 50,000 sequences, covering a high proportion of grape genes. 2,466 sequences have been obtained from a leaf cDNA library, and 2,438 from a berry library. Analysis of the leaf and berry EST's indicate 59% of clones have significant homology with known plant genes, 16% with more distant organisms and 25% may represent novel genes (no BLAST 2 match or match to EST's of unknown function). Redundancy levels in the leaf library are about 42%, and 39% in the berry library. Overall both libraries may yield more than 3,000 unique sequences. We are using expression in Arabidopsis to investigate the function of 58 EST's that match to 26 different transcription factors with unknown function in plants, and 330 EST's with no homology to known genes or other plant EST's. The inserts from the cDNA clones of interest are being cloned into pKMB and pSMB (1). Arabidopsis plants will be transformed with each construct to either over-express or knock out the cloned grape gene of interest. Transformed seeds will be germinated and the plants studied to determine phenotypic changes produced by the altered expression of the grape gene. The functional analysis in Arabidopsis of novel grape genes identified as EST's will provide a powerful new resource for discovering genes with the potential to improve table and wine grapes. (1) Mylne, J., and Botella, J.R. (1998) Binary Vectors for sense and antisense expression of Arabidopsis EST's. Plant Mol. Biol. Reporter 16, 257-262. Effie M. Ablett http://works.bepress.com/slade_lee/159 http://works.bepress.com/slade_lee/159 Sat, 31 Oct 2009 00:37:27 PDT Daniel LE Waters http://works.bepress.com/slade_lee/158 http://works.bepress.com/slade_lee/158 Sat, 31 Oct 2009 00:37:11 PDT Robert J. Henry http://works.bepress.com/slade_lee/157 http://works.bepress.com/slade_lee/157 Sat, 31 Oct 2009 00:36:56 PDT Margaret A. Wheeler