Hillary S. Sloane, James P. Landers, Kimberly A. Kelly

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Presentation transcript:

Hybridization-Induced Aggregation Technology for Practical Clinical Testing Hillary S. Sloane, James P. Landers, Kimberly A. Kelly The Journal of Molecular Diagnostics Volume 18, Issue 4, Pages 546-553 (July 2016) DOI: 10.1016/j.jmoldx.2016.02.004 Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Assay principles. Unpurified PCR product is added to a microwell containing probe-conjugated microbeads. A wild-type target hybridizes efficiently to both probes, inducing bead aggregation. Hybridization of a mutant target to the discriminating probe is unfavorable; therefore, the beads remain dispersed. An image of each microwell is obtained and processed to generate a saturation value corresponding to the extent of aggregation. The Journal of Molecular Diagnostics 2016 18, 546-553DOI: (10.1016/j.jmoldx.2016.02.004) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 KRAS mutation analysis of lung cancer and colorectal cancer cell lines. Using a PCR-derived target sequence, each cell line was analyzed using the HIAMD assay. The results indicate a significant decrease in the extent of aggregation produced by cell lines bearing a KRAS mutation (SW-620 and H2122) as compared to wild-type cell lines (CaCo2 and H1975). Data are expressed as means ± SD. n = 3 per group. The Journal of Molecular Diagnostics 2016 18, 546-553DOI: (10.1016/j.jmoldx.2016.02.004) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Resolution of mutant (mut) DNA in a background of wild-type (WT) DNA. HIAMD was applied for the analysis of synthetic samples with mixed genotypes. All samples with mutant content (down to 25% mutant) were significantly distinguishable from a wild-type sample. Data are expressed as means ± SD. n = 3 per group. The Journal of Molecular Diagnostics 2016 18, 546-553DOI: (10.1016/j.jmoldx.2016.02.004) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 KRAS mutation analysis of patient tissue samples. A: Representative sequencing results of three patient samples. (Note: the reverse strand was sequenced). B: HIAMD results. Aggregation of 36% was used as the threshold value to distinguish between wild-type (≥36%) and mutant (<36%) genotypes. Patient numbers are displayed on the x axis. Data are expressed as means ± SD. The Journal of Molecular Diagnostics 2016 18, 546-553DOI: (10.1016/j.jmoldx.2016.02.004) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions