Danielle C. Smith, Alina Esterhuizen, Jacquie Greenberg

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Presentation transcript:

Caution Regarding the Interpretation of Homoallelism in Polyglutamine Multiplex Assays Danielle C. Smith, Alina Esterhuizen, Jacquie Greenberg The Journal of Molecular Diagnostics Volume 15, Issue 5, Pages 706-709 (September 2013) DOI: 10.1016/j.jmoldx.2013.05.009 Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Sequencing of the multiplex primer-binding regions in ATXN7. Sequencing chromatograms of three representative samples (A–C), showing no evident polymorphisms within the binding regions of the multiplex primers (forward and reverse). The Journal of Molecular Diagnostics 2013 15, 706-709DOI: (10.1016/j.jmoldx.2013.05.009) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Positions of primer binding around the ATXN7 CAG repeat. The relative positions of primer-binding sites of the sequencing primers, multiplex primers,4 and singleplex primers are shown. Sequencing and singleplex primers were designed in house. The Journal of Molecular Diagnostics 2013 15, 706-709DOI: (10.1016/j.jmoldx.2013.05.009) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Assay design and comparison of results obtained from conventional end-labeled PCR and TP-PCR. A: In end-labeled PCR assays, two primers are designed to flank the CAG repeat region (one primer is fluorescently labeled). The electropherogram shows a single peak corresponding to the wild-type allele, along with multiple peaks indicating the unstable expanded allele. B: TP-PCR assays contain a single labeled flanking primer, along with a repeat-specific primer that binds to the expansion. A third primer binds to a tail region of the repeat-specific primer. The electropherogram shows a ladder-like pattern resulting from the repeat-specific primer binding to the CAG repeat region at multiple points. Stars indicate fluorescent labels. The Journal of Molecular Diagnostics 2013 15, 706-709DOI: (10.1016/j.jmoldx.2013.05.009) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions