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Defining the performance parameters of a rapid screening tool for myotonic dystrophy type 1 based on triplet-primed PCR and melt curve analysis |
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| Authors: | Mulias Lian Hai-Yang Law Caroline G. Lee |
| Affiliation: | 1. Khoo Teck Puat – National University Children’s Medical Institute, National University Health System, Singapore, Singapore;2. Department of Pediatric Medicine, KK Women’s and Children’s Hospital, Singapore, Singapore;3. Pediatrics Academic Clinical Program, Duke-NUS Graduate Medical School, Singapore, Singapore;4. Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore;5. Cancer and Stem Cell Biology Program, Duke-NUS Graduate Medical School, Singapore, Singapore;6. Division of Medical Sciences, National Cancer Center, Singapore, Singapore |
| Abstract: | Background: DMPK CTG-repeat expansions that cause myotonic dystrophy type 1 (DM1) can be detected more rapidly, cost-effectively, and simply by combining triplet-primed PCR (TP-PCR) with melting curve analysis (MCA). We undertook a detailed technical validation study to define the optimal operational parameters for performing bidirectional TP-PCR MCA assays. Methods: We determined the assays’ analytic specificity and sensitivity, assessed the effect of reaction volumes, DNA diluents, and common contaminants on melt peak temperature, determined the assays’ sensitivity in detecting low-level mosaicism for repeat expansion, and evaluated their performance on two real-time PCR platforms. Results: Both assays were highly specific and sensitive, and performed optimally under a broad range of parameters. Bidirectional TP-PCR MCA analysis also reduces the risk of generating false-negative results associated with the rare CCG-interruptions that may be present at either end of expanded alleles. Conclusion: The DMPK TP-PCR MCA is a highly specific, sensitive, and significantly cost-saving screening tool for DM1. |
| Keywords: | Myotonic dystrophy type 1 DMPK TP-PCR MCA screening |