Novel Heat Pulse Extension-PCR–Based Method for Detection of Large CTG-Repeat Expansions in Myotonic Dystrophy Type 1 Arto K. Orpana, Tho H. Ho, Katariina.

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Presentation transcript:

Novel Heat Pulse Extension-PCR–Based Method for Detection of Large CTG-Repeat Expansions in Myotonic Dystrophy Type 1 Arto K. Orpana, Tho H. Ho, Katariina Alagrund, Maaret Ridanpää, Kristiina Aittomäki, Jakob Stenman The Journal of Molecular Diagnostics Volume 15, Issue 1, Pages 110-115 (January 2013) DOI: 10.1016/j.jmoldx.2012.07.004 Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Illustrative temperature profile for each cycle of the HPE-PCR cycling protocol. After initial denaturation, 40 cycles of PCR amplification were performed with a two-stage denaturation step (95°C for 45 seconds and 98°C for 10 seconds), annealing at 68°C for 30 seconds, followed by gradual heating to a basal extension temperature of 76°C (30% ramp rate) and 21 heat pulses with a peak temperature of 83°C during the extension step. The Journal of Molecular Diagnostics 2013 15, 110-115DOI: (10.1016/j.jmoldx.2012.07.004) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Amplification over CTG-repeat loci from a panel of 18 DM1 patients and one healthy individual using HPE-PCR. A: Agarose gel was stained with ethidium bromide. Samples were selected based on the repeat expansion size originally found in the Southern blot analysis. Severe tissue heterogeneity in several samples (asterisks) could also be reliably detected. B: Negative image of A, showing improved contrast for some samples. Patient identification numbers shown on top were used throughout this article. The Journal of Molecular Diagnostics 2013 15, 110-115DOI: (10.1016/j.jmoldx.2012.07.004) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Concordance between Southern blot and HPE-PCR results. Southern blot (A) and corresponding HPE-PCR (B) analysis of DNA samples from a healthy individual (WT), a patient with congenital DM1 (C) and a patient with adult-onset DM1 (A). The expanded alleles in the congenital DM1 sample were amplified with high efficiency despite the short wild-type allele. Tissue heterogeneity and variation of the expansion fragment length in the adult-onset DM1 sample, seen on Southern blot analysis, is well reproduced by HPE-PCR. The Journal of Molecular Diagnostics 2013 15, 110-115DOI: (10.1016/j.jmoldx.2012.07.004) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions