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Abstract

BioTechniquesVol. 46, No. 7 BioSpotlightOpen AccessBioSpotlightPatrick C.H. Lo & Kristie NyboPatrick C.H. LoSearch for more papers by this author & Kristie NyboSearch for more papers by this authorPublished Online:25 Apr 2018https://doi.org/10.2144/000113174AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail FRET-ting about Taqman probesMultiplexed real-time PCR reactions using hybridization-specific probes in real-time PCR machines equipped with a single emission channel and multiple detection channels are not possible with standard Taqman probes and require the use of FRET (fluorescence resonance energy transfer) probes. FRET probes, however, may be less robust in suboptimal conditions than Taqman probes and can be difficult to design due to the need to identify a longer sequence that can accommodate the binding of the two required probes: the donor fluorophore–containing probe which hybridizes adjacent to the probe with the acceptor fluorophore. As an alternative, P. Jothikumar, V. Hill, and J. Naranyanan at the Georgia Institute of Technology (Atlanta, GA, USA) have developed a novel triple-labeled Taqman probe that can be used in conjunction with a standard Taqman probe to easily duplex real-time PCR reactions in single-emission-channel real-time PCR machines. Both Taqman probes they used have an appropriate BHQ quencher at its 3′ end, but whereas the standard probe has a FAM fluorophore at its 5′ end, their so-called “FRET-Taqman” probe has both FAM and Cy5.5 at its 5′ end. After hybridization to their respective targets and polymerase-mediated hydrolysis of the probes in a duplexed reaction that is carried out in a cycler with a single blue LED excitation source (at 470 nm), emission fluorescence from FAM in the standard Taqman probe is at 520 nm, while in the FRET-Taqman probe, complete transfer of the emission energy from the excited FAM to Cy5.5 through FRET results in fluorescence only at 705 nm from Cy5.5.See “Design of FRET-taqman probes for multiplex real-time PCR using an internal positive control”Restricting Parental InterferenceTechniques for site-directed mutagenesis (SDM) typically depend on the extension of oligonucleotide primers that are complementary to the target sequence and also carry the desired point mutation. Existing PCR- and non-PCR–based SDM methods can be limited by amplicon length, replication fidelity, and their small spectrum of possible mutations. Plasmid recombineering with the Red/ET system, which uses 50-base-long tails on the donor DNA for homologous recombination into target DNA, would be a superior methodology for SDM since it allows for the sequence- and size-independent manipulation of DNA with multiple simultaneous sequence modifications, but it suffers several drawbacks: multimer formation, low efficiency which necessitates extensive colony screening, and difficult counter-selection caused by contaminating parental plasmids in multicopy plasmid situations. S. Noll, H. Kranz, and colleagues at Gene Bridges GmbH (Heidelberg, Germany) have combined unique restriction site elimination (USE) with recombineering in a method that overcomes these problems. They PCR-amplified drug-resistance cassettes with primers containing the 50-bp regions flanking the target site for homologous recombination and a restriction enzyme linearization site not present in the plasmid. The product was then co-electroporated with the target plasmid into Red/ ET-proficient Escherichia coli cells and the resulting plasmid, with the drug-resistance marker, was isolated. In a second recombineering step, linear DNA with the desired mutation replaced the drug-resistance cassette at the target site in the recombinant plasmids. This step also eliminated the introduced restriction site, allowing for selective linearization of parental plasmids. Eliminating the ability of the parental plasmid to transform E. coli greatly improved the efficiency of isolating the mutated plasmid.See “Site-directed mutagenesis of multi copy-number plasmids: Red/ET recombination and unique restriction site elimination”FiguresReferencesRelatedDetails Vol. 46, No. 7 Follow us on social media for the latest updates Metrics History Published online 25 April 2018 Published in print June 2009 Information© 2009 Author(s)PDF download