Design and Validation of a Conformation Sensitive Capillary Electrophoresis-Based Mutation Scanning System and Automated Data Analysis of the More than.

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Presentation transcript:

Design and Validation of a Conformation Sensitive Capillary Electrophoresis-Based Mutation Scanning System and Automated Data Analysis of the More than 15 kbp- Spanning Coding Sequence of the SACS Gene Sascha Vermeer, Rowdy P.P. Meijer, Tom G.J. Hofste, Daniëlle Bodmer, Ermanno A.J. Bosgoed, Frans P.M. Cremers, Berry H.P. Kremer, Nine V.A.M. Knoers, Hans Scheffer The Journal of Molecular Diagnostics Volume 11, Issue 6, Pages 514-523 (November 2009) DOI: 10.2353/jmoldx.2009.090059 Copyright © 2009 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Example of initially false negative capillary heteroduplex electropherogram. This electropherogram shows the analysis of amplicon 11 containing the heterozygous SACS c.2080G>A variation, indicated by the arrow. The pattern of the sample containing the variation and the pattern of the normal genotype sample are virtually identical. This variation is located at nucleotide 151 of the 488 nucleotides in total. This variation could be detected after optimization of the threshold values of the data analysis software (Bionumerics). This underlines that optimization of the data analysis program is crucial. The Journal of Molecular Diagnostics 2009 11, 514-523DOI: (10.2353/jmoldx.2009.090059) Copyright © 2009 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 Example of false positive capillary heteroduplex electropherogram. This electropherogram shows the true false-positive result in amplicon 38 (exon 9). The Journal of Molecular Diagnostics 2009 11, 514-523DOI: (10.2353/jmoldx.2009.090059) Copyright © 2009 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Example of a variation in two overlapping fragments. Due to overlap in the amplicons, five variations have been detected in two consecutive amplicons (Tables 2and 3). Figure 3 shows the mutation (indicated by the arrow) c.1475G>A in amplicon 9 (located at nucleotide 448 of the 484 nucleotides in total) and 10 (at nucleotide 84 of the 449 in total). The Journal of Molecular Diagnostics 2009 11, 514-523DOI: (10.2353/jmoldx.2009.090059) Copyright © 2009 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 Example of two variations in a single amplicon. In three samples, two variations per amplicon (15, 35, and 41 [exon 9]) were present, resulting in a pattern of multiple conformational changes. For example, panel A (amplicon 41 [c.12160C>T and c.12304T>C]) shows multiple conformational changes as the variants are located on different alleles. The separate patterns when occurring as a single variation (B) are displayed beneath for comparison. The arrows indicate the variations. The Journal of Molecular Diagnostics 2009 11, 514-523DOI: (10.2353/jmoldx.2009.090059) Copyright © 2009 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions