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Genetics 179, 177-192. The small ubiquitin-like modifier (SUMO) and SUMO-conjugating system of Chlamydomonas reinhardtii. 2008

Wang, Y., Ladunga, I., Miller, A.R., Horken, K.M., Plucinak, T., Weeks, D.P. and Bailey, C.P.

Notes: These authors used computational biology to screen the genome of the alga Chlamydomonas reinhardtii for SUMO (small ubiquitin-like modifier) homologs. They identified several SUMO and SUMO-like sequences. One of these proteins, crSUMO96, which was recognized by the A. thaliana anti-SUMO antibody, was studied in detail. During their studies, the authors used the PureYield™ RNA Midiprep System to isolate total RNA from C. reinhardtii cells. This RNA was used in real-time RT-RCR assays to detect mRNA transcripts for the various SUMO-like proteins. The Plexor® Two-Step qRT-PCR System was used for the real-time assays. For expression studies, cDNA encoding the various proteins was amplified and subcloned into the pGEM®-T Easy Vector before transfer into an expression vector. (3875)

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Phytopathology 97, 865-872. Multiplex real-time quantitative PCR to detect and quantify Verticillium dahliae colonization in potato lines that differ in response to Verticillium wilt. 2007

Atallah, Z.K., Bae, J., Jansky, S.H., Rouse, D.I., and Stevenson, W.R.

Notes: These authors developed a quantitative, real-time PCR method for the detection of Verticillium dahliae in potato cultivars. V. dahiae is the causative agent of Verticillium wilt, also known as potato early dying (PED). The standard detection method is a plating assay that takes 2 weeks to complete. The authors of this study performed qPCR assays using the V. dahliae beta tubulin-2 gene as a target. Initially, they amplified, subcloned and sequenced seven different genes in order to identify targets that were polymorphic among the genus Verticillium, but monoprphic in V. dahliae. After selection of beta-tubulin 2 as a suitable target, monoplex and duplex qPCR assays were performed using a Bio-Rad iCycler thermal cycler and 1ng DNA from various cultivars. For the duplex assays, the Plexor® qPCR System was used to amplify both beta-tubulin 2 and beta-actin genes simultaneously. One beta-tubulin primer was labeled with FAM, and one actin primer was labeled with Redmond Red phopsphoramidite. Amplifications were performed in 25µl reactions with 200nM each primer, 1ng DNA, and the Plexor® Master Mix. Cycling conditions were as follows: 2 minutes at 95°C; 40 cycles of 5s at 95°C, 35s at 61°C. Melt curve analysis was performed to confirm the specificity of the amplification products. The qPCR assays were shown to be faster and more sensitive than the standard plating technique, and were one order of magnitude more sensitive than other PCR-based assays. (3673)

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Hum. Mutat. 0, 1-6. Novel Plexor SNP genotyping technology: comparisons with TaqMan and homogenous MassEXTEND MALDI-TOF mass spectrometry. 2007

Tindall, E.A., Speight, G., Petersen, D.C., Padilla, E.J., and Hayes, V.M.

Notes: This study compared the performance of the Plexor® qPCR System with the TaqMan® and MassEXTEND™ methods for genotyping analysis of 11 SNPs in >2000 DNA samples. All three methods were shown to be equivalent in call rate and accuracy. The Plexor® System is described as a cost-effective, efficient alternative to the TaqMan® technology for medium-throughput SNP analysis. Plexor® qPCR System reactions contained 5ng template DNA, 0.2µl 5µM allele-specific primers, 0.2µl 10µM anchor primer, and 2.5µl 2X Plexor™ Master Mix in a total volume of 5µl. PCR was performed on an ABI PRISM® 7900HT Sequence Detection System using the following cycling conditions: 95°C for 2 minutes; 50°C for 35s; and 40 cycles of 95°C for 5s, 60°C for 35s. Primers were designed using the Plexor® Primer Design Software. (3674)

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