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.
| Frumkin, Jesse P; Patra, Biranchi N; Sevold, Anthony et al. (2016) The interplay between chromosome stability and cell cycle control explored through gene-gene interaction and computational simulation. Nucleic Acids Res 44:8073-85 |
| Johnson, Leah M; Gao, Lu; Shields IV, C Wyatt et al. (2013) Elastomeric microparticles for acoustic mediated bioseparations. J Nanobiotechnology 11:22 |
| Micheva-Viteva, Sofiya N; Shou, Yulin; Nowak-Lovato, Kristy L et al. (2013) c-KIT signaling is targeted by pathogenic Yersinia to suppress the host immune response. BMC Microbiol 13:249 |
| Ai, Ye; Sanders, Claire K; Marrone, Babetta L (2013) Separation of Escherichia coli bacteria from peripheral blood mononuclear cells using standing surface acoustic waves. Anal Chem 85:9126-34 |
| Sanders, Claire K; Mourant, Judith R (2013) Advantages of full spectrum flow cytometry. J Biomed Opt 18:037004 |
| Cushing, Kevin W; Piyasena, Menake E; Carroll, Nick J et al. (2013) Elastomeric negative acoustic contrast particles for affinity capture assays. Anal Chem 85:2208-15 |
| 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 |
| Marina, Oana C; Sanders, Claire K; Mourant, Judith R (2012) Correlating light scattering with internal cellular structures. Biomed Opt Express 3:296-312 |
| Houston, Jessica P; Naivar, Mark A; Jenkins, Patrick et al. (2012) Capture of Fluorescence Decay Times by Flow Cytometry. Curr Protoc Cytom 59:1.25.1-1.25.21 |
| Marina, Oana C; Sanders, Claire K; Mourant, Judith R (2012) Effects of acetic acid on light scattering from cells. J Biomed Opt 17:085002-1 |
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