Session Title: Genomics   Session Type: Poster

Session Location: Exhibit Hall   Session Time: Wed 4:30PM-6:30PM, Thu 4:30PM-6:30PM, Fri 1:30PM-3:30PM

Abstract Information

Poster Board Number: 1273/F   Presentation Time: Fri, Oct. 28, 2005, 1:30PM-3:30PM

Keywords: KW013 - characterization of disorders, KW057 - genomic methodologies, KW079 - methodology, KW086 - mutation detection, KW009 - cancer cytogenetics

Abstract Content

Genomic Copy Number Detection with Microsphere-Based Suspension Hybridization. H.L. Newkirk1, 2, M. Miralles1, 2, P.K. Rogan1, 2, J.H. Knoll1, 2. 1) Children’s Mercy Hospitals and Clinics School of Medicine Children’s Mercy Hospitals and Clinics; 2) School of Medicine University of Missouri-Kansas City.

   A microsphere-based suspension hybridization assay has been developed for high throughput detection of changes in genomic copy number. This assay allows for the direct analysis of whole genomic DNA extracted from residual fixed cell pellets and nick translated with biotin-dUTP. Single copy (sc) probes ranging in length from 60 to 2300 bp (100 bp average; Rogan et al Genome Res 11:1086, 2001) from ABL1 (chromosome 9q34), TEKT3 (17p12) and HOXB1 (7p15) were conjugated to spectrally-distinct polystyrene microspheres (Molecular Probes). Reactions were hybridized and detected using streptavidin-phycoerythrin conjugate and analyzed by flow cytometry. Using multiplex hybridization assays, 5’ ABL1 deletions in patients with chronic myelogenous leukemia were confirmed by comparison of mean fluorescence intensities of chromosome 9q34 probes with the HOXB1 control (2 copies per diploid genome). The intensities of the chromosome 9 probes were significantly reduced in 5 deletion patients relative to the control probe. Similar experiments were performed with three trisomic 9 cell lines and in four duplication positive patients with Charcot-Marie-Tooth Type 1a disease (CMT1a). Using HOXB1 as an internal control, the assay distinguished 3 vs 2 alleles of chromosome 9 and 17 probes in the abnormal cell lines, where the mean fluorescence intensity of the chromosome 9 and 17 probes was about one third higher than for the control probe. Reproducibility studies for all patient samples yielded mean fluorescence intensity ratios that consistently distinguished copy number differences between the control probe and ABL1 or CMT1a probes. Unlike oligonucleotide bead hybridization assays, prior amplification of locus-specific target DNA was not required because the increased length and single copy composition of the hybridization probes increase the specificity required for direct detection of homologous genomic target sequences.