Improved Assays for AGG Interruptions in Fragile X Premutation Carriers  Bruce E. Hayward, Karen Usdin  The Journal of Molecular Diagnostics  Volume 19, Issue 6, Pages 828-835 (November 2017) DOI: 10.1016/j.jmoldx.2017.06.008 Copyright © 2017 Terms and Conditions

Figure 1 Map of the 5′ end of the FMR1 premutation (PM) allele in the HT51 cell line showing the location of the enzymes used for cloning and the primer binding sites used for the various PCR assays described. The positions of the two AGG interruptions present in this allele are indicated by red lines. The primer binding sites of the forward primers used in the new assays are indicated by blue arrows and the reverse primers by pink arrows. The Journal of Molecular Diagnostics 2017 19, 828-835DOI: (10.1016/j.jmoldx.2017.06.008) Copyright © 2017 Terms and Conditions

Figure 2 Diagrammatic representation of the principle behind the different triplet-primed assays for AGG interruptions. The principle of the previously published assays (A) and the new generation of assays described here (B).8,9 The arrows represent the binding sites for the primer used. Asterisks indicate the labeled primer for each reaction, Not_FraxR4 for the CGG-primed reaction and Not_FraxC for the CCG-primed reaction, respectively. The dotted arrow in A illustrates the second flanking primer used in these reactions, Not_FraxC and Not_FraxR4, respectively. Some of the primer binding sites in the CCG-primed assays are shown in gray because smaller PCR products are favored in these reactions; thus, the products resulting from priming at these sites are only a minor fraction of the PCR products produced. The gray parallelograms in A illustrate the location of the region of the repeat tract where priming does not occur and corresponds to the dips seen in the resulting electrophoretograms. PM, permutation. The Journal of Molecular Diagnostics 2017 19, 828-835DOI: (10.1016/j.jmoldx.2017.06.008) Copyright © 2017 Terms and Conditions

Figure 3 CGG-primed (A) and CCG-primed (B) PCR assays for C0366, HT51A, and SC120A. PCR amplification of the three genomic templates was performed as described in the Materials and Methods, and the products were resolved by capillary electrophoresis. The insert shown in each electrophoretogram is an enlargement of the 300- to 700-bp region (A). The PCR products corresponding to the full-length normal allele in the permutation (PM) females are indicated by the open arrowhead and the location of full-length PM alleles by the closed arrowhead. The lack of PCR products resulting from failure to prime at interruptions on the PM alleles are shown by the black arrows and those on the normal allele by the gray arrows. RFU, relative fluorescent units. The Journal of Molecular Diagnostics 2017 19, 828-835DOI: (10.1016/j.jmoldx.2017.06.008) Copyright © 2017 Terms and Conditions

Figure 4 A-primed (A) and T-primed (B) PCR assays for C0366, HT51A, and SC120A. PCR amplification of the three genomic templates was performed as described in Materials and Methods using HEX-labeled Not_FraxR4 and the A-primer for the A-primed reaction and FAM-labeled Not_FraxC and the T-primer for the T-primed reactions. The products were then resolved by capillary electrophoresis. The products arising from priming at AGG interruptions are marked with the letter A (black font for normal alleles, red font for PM alleles). The positions of interruptions in the T-primed reaction are marked with the letter T. RFU, relative fluorescent units. The Journal of Molecular Diagnostics 2017 19, 828-835DOI: (10.1016/j.jmoldx.2017.06.008) Copyright © 2017 Terms and Conditions

Figure 5 Repeat assay and A-primed assays for C0223, a premutation (PM) female with a heterogeneous mixture of allele sizes. PCR amplification of the genomic DNA (gDNA) from C0233 was performed as described in the Materials and Methods using HEX-labeled Not_FraxR4 and Not_FraxC for the repeat number assay and HEX-labeled Not_FraxR4 and the A-primer for the A-primed assay. The products were then resolved by capillary electrophoresis. The inset in each case shows an expanded version of the electrophoretogram. The products corresponding to the full-length alleles are indicated by the open (normal) and closed (PM) arrowheads. The products arising from priming at AGG interruptions are marked with the letter A (black font fornormal alleles, red for PM alleles). The bracket in the repeat number assay indicates the somatic expansions present in this individual. RFU, relative fluorescent units. The Journal of Molecular Diagnostics 2017 19, 828-835DOI: (10.1016/j.jmoldx.2017.06.008) Copyright © 2017 Terms and Conditions

Figure 6 A-primed PCR assays for C0366 and SC120A in the presence of the internal PCR control template. PCR amplification of the genomic templates was performed in the presence of an equal molar amount of the internal control fragment as described in Materials and Methods using HEX-labeled Not_FraxR4, NED-labeled Not_PsdR4, and the unlabeled A-primer. The products were then resolved by capillary electrophoresis. The top panels show the reaction products detected in the HEX channel [genomic DNA (gDNA) products] and the bottom panels show the reaction products detected in the NED channel (the internal control products). The products arising from priming at AGG interruptions are marked with the letter A (black font for normal alleles, red for PM alleles). Note that the minor peaks seen in the NED channel are bleed-through/pull-up peaks from the ROX channel (containing the MapMarker 1000-ROX ladder) and thus indicate the positions of the individual fragments in the molecular weight ladder. PM, permutation; RFU, relative fluorescent units. The Journal of Molecular Diagnostics 2017 19, 828-835DOI: (10.1016/j.jmoldx.2017.06.008) Copyright © 2017 Terms and Conditions