omation mbers - Society for Laboratory Automation and Screening

11:00 am Wednesday, February 4 Genomics – SNPs Room A1
Robert B. Cary
Los Alamos National Laboratory
P.O. Box 1663 MS M888
Los Alamos, New Mexico 87545
rbcary@lanl.gov
100
Co-Author(s)
Hong Cai
Richard T. Okinaka
Paige E. Pardington
Sensitive Detection and Identification of Threat Agents by Microarray-based Genotyping
Using MLST Derived Signatures
The rapid, accurate and sensitive detection of biological warfare agents requires a broad-spectrum assays capable
of discriminating closely related microbial and viral pathogens. Moreover, in those cases where a biological agent
release has been identified, forensic analysis demands detailed genetic signature data for accurate subspecies
identification and attribution. Identification as well as the determination of important phenotypes, such as antibiotic
resistance, can be accomplished by the examination of single nucleotide polymorphisms (SNPs) across multiple
genetic loci. We have adapted a simple microarray-based genotyping assay to the detection of microbial pathogen
signatures derived from multi-locus sequence typing (MLST) analyses. Using SNP signatures derived from MLST
analysis of strains of Bacillus anthracis and its near neighbors we demonstrate that the assay can be used as a
sensitive method for the detection and identification of Bacilli derived DNA while simultaneously providing accurate
and detailed genotype information useful for subspecies identification. This work was funded by United States
Department of Energy, CBNP funding to RBC. LA-UR-03-3343.
11:30 am Wednesday, February 4 Genomics – SNPs Room A1
Ming Xiao
University of California, San Francisco
Cardiovascular Research Institute
505 Parnassus Avenue, Long 1329, Box 0130
San Francisco, California 94143-0130
mxiao@itsa.ucsf.edu
Improvements on the TDI-FP SNP Genotyping Assay
The TDI-FP (the template-directed dye-terminator incorporation assay with fluorescence polarization detection) is
a homogeneous SNP genotyping assay. In the FP-TDI assay, the allele-specific dye terminators are incorporated
onto an unlabeled SNP specific primer. The genotypes are inferred by the FP increase of dye terminators. FP
detection works best when the reaction is driven into completion. The misincorporation of dye-terminators and
the differential incorporation efficiency of two dye-terminators are the major reasons for the failure of the assay.
We have optimized the assay by choosing two dye-terminators with equal incorporation efficiency and limiting the
misincorporation.