Efficient and Highly Sensitive Screen for Myotonic Dystrophy Type 1 Using a One-Step Triplet-Primed PCR and Melting Curve Assay  Mulias Lian, Indhu-Shree Rajan-Babu, Kunal Singh, Caroline G. Lee, Hai-Yang Law, Samuel S. Chong  The Journal of Molecular Diagnostics  Volume 17, Issue 2, Pages 128-135 (March 2015) DOI: 10.1016/j.jmoldx.2014.10.001 Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 5′ and 3′ triplet-primed PCR (TP-PCR) at the DMPK CTG repeat locus. A: Schematic illustration of 5′ and 3′ TP-PCRs showing positions of primers. B: Melt peak profiles and corresponding electropherograms of 5′ and 3′ TP-PCR amplicons from six myotonic dystrophy type 1-unaffected Coriell Cell Repositories samples. Both 5′ and 3′ TP-PCR products generate melt peaks with Tms ranging from 81.2°C to 81.75°C. -dF/dT, negative first derivative of fluorescence versus temperature; RFU, relative fluorescence unit. The Journal of Molecular Diagnostics 2015 17, 128-135DOI: (10.1016/j.jmoldx.2014.10.001) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Melt peaks and corresponding electropherograms of 5′ and 3′ triplet-primed PCR (TP-PCR) amplicons from nine myotonic dystrophy type 1-affected Coriell Cell Repositories (CCR) samples. Sample IDs and allele sizes provided by CCR are shown. Insets show higher resolution of longer TP-PCR products. Both 5′ and 3′ TP-PCR products generate melt peaks with Tms exceeding 85°C. Tms are generally positively correlated with repeat size. -dF/dT, negative first derivative of fluorescence versus temperature; RFU, relative fluorescence unit. The Journal of Molecular Diagnostics 2015 17, 128-135DOI: (10.1016/j.jmoldx.2014.10.001) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 A and B: Normalized melt curves (A) and melt peaks (B) of the 5′ and 3′ triplet-primed PCR (TP-PCR) amplicons from 17 Coriell Cell Repositories samples. Melt peak threshold temperatures (TTs) are indicated by vertical dotted lines in B. Melt curve analysis separate unaffected and myotonic dystrophy type 1–affected samples to the left and right of the TTs established by pDMPK(CTG)35 and pDMPK(CTG)48, respectively, in either melt curve or melt peak display format. C: Electropherograms of pDMPK(CTG)35 and pDMPK(CTG)48. -dF/dT, negative first derivative of fluorescence versus temperature; RFU, relative fluorescence unit. The Journal of Molecular Diagnostics 2015 17, 128-135DOI: (10.1016/j.jmoldx.2014.10.001) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Normalized melt curves (A), melt peaks (B), and selected electropherograms (C) of 5′ and 3′ triplet-primed PCR (TP-PCR) amplicons from 60 clinical samples. Both 5′ and 3′ TP-PCR melt curve analysis (MCA) assays accurately identified the 35 unaffected and 25 myotonic dystrophy type 1–affected samples. MCA and GeneScan electropherogram results are completely concordant. The Journal of Molecular Diagnostics 2015 17, 128-135DOI: (10.1016/j.jmoldx.2014.10.001) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 5 A: Melt peak profiles and corresponding electropherograms (insets) of 5′ and 3′ triplet-primed PCR (TP-PCR) amplicons from peripheral blood–, buccal swab–, and saliva-extracted genomic DNA of two volunteers. All samples were assayed in triplicate. Melt peak temperatures (Tms) of each sample type are shown in brackets. Both 5′ and 3′ TP-PCR melting curve analyses amplify successfully from genomic DNA extracted from buccal swabs and saliva, and generate Tms comparable to that from peripheral blood and concordant with their DMPK genotypes. B: Melt peaks of 5′ and 3′ TP-PCR amplicons from 1 ng, 10 ng, 50 ng, and 100 ng of GM16206 (12 and 14 CTGs) and GM05164 (21 and approximately 340 CTGs) genomic DNA. Tms are shown in brackets. DNA quantity as low as 10 ng generates a distinct melt peak. -dF/dT, negative first derivative of fluorescence versus temperature; RFU, relative fluorescence unit. The Journal of Molecular Diagnostics 2015 17, 128-135DOI: (10.1016/j.jmoldx.2014.10.001) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 6 Frequency distribution of CTG repeat alleles in normal individuals from European, Japanese, African American, Indian, African Negroid, and Chinese populations. The largest normal DMPK allele identified, from 2190 chromosomes screened, was 35 CTG repeats in the European and Japanese populations. The Journal of Molecular Diagnostics 2015 17, 128-135DOI: (10.1016/j.jmoldx.2014.10.001) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions