This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Analysis of DNA sequence variation is important for identifying disease related genes and diagnosing disease susceptibility. The most common type of genetic variation is the single nucleotide polymorphism (SNP), which may occur as frequently as 1 in every 1000 DNA bases. The goal of this project is to develop, validate, and demonstrate new approaches to the discovery and scoring of single nucleotide polymorphisms (SNPs). The new methods will take advantage of versatile and widely available measurement platform, flow cytometry, to provide rapid and sensitive sample analysis without wash steps. De novo SNP detection will be achieved using an immobilized mismatch binding protein to bind fluorescently labeled heteroduplex DNA to microspheres, which will then be analyzed by flow cytometry. We will evaluate different mismatch binding proteins, immobilization approaches, and labeling strategies to develop a homogeneous assay compatible with multiplexed analysis by flow cytometry. The optimized assay will be sensitive, rapid, and scaleable to scan amplified genomic sequence for SNPs at rates of greater than 1 Mb/day. SNP scoring will be performed using microsphere-based minisequencing in which primers will be extended with fluorescent dideoxynucleotides using polymerase, and then captured on beads and analyzed by flow cytometry. By employing a unique scheme of capture sequences to address differently dyed microspheres, we will be able to score simultaneously dozens, and potentially hundreds, of SNPs from a single genomic sample in 1-2 minutes. We will validate and demonstrate these new approaches in collaboration with laboratories currently using conventional technologies to discover and score SNPs for use in disease diagnostics, pharmacogenetics, and mapping and linkage studies. Both our SNP discovery and scoring methods will be compatible commercial flow cytometers present in most universities, research institutes, and clinical diagnostic laboratories.
| 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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