As one of our lab specializations of the DOE Joint Genome Institute (JGI), simple sequence polymorphism (SSP) detection is part of the lead LANL is expected to take in the area of functional genomics. Any R&D efforts to improve the detection of polymorphisms will help achieve goals of higher thoughput and denser coverage for the placement of SSPs on the physical map. Current work at the National Flow Cytometry Resource at LANL is focused on the development of quantitative, robust methods for the analysis of protein-nucleic acid and nucleic acid-nucleic acid interactions, as well as the development of automated sample handling suitable for high-throughput screening methods. One of the goals of the present project is the development of flow cytometric approaches to the detection and characterization of polymorphisms. Once the locations of polymorphisms are identified, the frequency of each nucleotide (A, C, T, G) incorporation at the site may be measured using a fluorescent dideoxynucleotide extension of targeted primers. In one scenario, primers targeted to a specific site are attached to a microsphere, and genomic DNA is allowed to hybridize. The primer is extended by polymerase to incorporate one of four dideoxy nucleotides, each labeled with a different fluorophore. The intensity of fluorescence from each of the labeled nucleotides on the beads is then measured by flow cytometry. This approach could be multiplexed by attaching unique primers to different size beads, enabling the simultaneous measurement of frequencies at a number of different loci. It is expected that the application of flow cytometric technologies can provide methods useful for rapidly detecting and analyzing single nucleotide substitutions. Their use in medical research and functional genomics will help a great deal in the discovery and development of treatments for many human diseases, and will also provide tools to investigate evolutionary histories with greater resolution than currently possible.
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| Piyasena, Menake E; Austin Suthanthiraraj, Pearlson P; Applegate Jr, Robert W et al. (2012) Multinode acoustic focusing for parallel flow cytometry. Anal Chem 84:1831-9 |
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| Vuyisich, Momchilo; Sanders, Claire K; Graves, Steven W (2012) Binding and cell intoxication studies of anthrax lethal toxin. Mol Biol Rep 39:5897-903 |
| Chaudhary, Anu; Ganguly, Kumkum; Cabantous, Stephanie et al. (2012) The Brucella TIR-like protein TcpB interacts with the death domain of MyD88. Biochem Biophys Res Commun 417:299-304 |
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