Nucleotide Extension Genotyping by High-Resolution Melting Michael Liew, Carl Wittwer, Karl V. Voelkerding  The Journal of Molecular Diagnostics  Volume 12, Issue 6, Pages 731-738 (November 2010) DOI: 10.2353/jmoldx.2010.090149 Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Schematic diagram of the SNE procedure. A: SNE components are separated from PCR components by placement in the cap of the LightCycler capillary. The reactions are then subjected to PCR thermal cycling. B: Post-PCR, capillaries are centrifuged to mix the components together followed by SNE thermal cycling. Examples of extension products for the RET 1853G>A SNP target are shown using each of the ddNTPs as well as EDTA as a no extension control. The lengths of the extension products are determined by the template sequence. The theoretical melting temperature (Tm) of each extension product is also displayed. C: Shown are locations of the primers (gray text), SNE oligonucleotide (underlined text), and SNP (highlighted text) for the RET c.1853G>A target. The Journal of Molecular Diagnostics 2010 12, 731-738DOI: (10.2353/jmoldx.2010.090149) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 SNE of RET c.1853 homozygous variants. Each panel shows a different homozygous variant, G/G (A), A/A (B), C/C (C), and T/T (D). Melting curves generated by the different ddNTPs and no extension controls are labeled in each panel. ddATP = thin black solid line, ddTTP = thin gray dashed line, ddCTP = thin black dashed line, ddGTP = thick gray solid line, and no extension control = thick black solid line. The Journal of Molecular Diagnostics 2010 12, 731-738DOI: (10.2353/jmoldx.2010.090149) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 SNE of RET c.1853 heterozygous variants. Heterozygous A/G (A), T/G (B), and C/G (C) are shown. Melting curves generated by the different ddNTPs and no extension controls are labeled in each panel. ddATP = thin black solid line, ddTTP = thin gray dashed line, ddCTP = thin black dashed line, ddGTP = thick gray solid line, and no extension control = thick black solid line. The Journal of Molecular Diagnostics 2010 12, 731-738DOI: (10.2353/jmoldx.2010.090149) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 SNE of HFE c.187 using a 22-mer extension oligonucleotide. Wild-type C/C (A), heterozygous C/G (B), and mutant G/G (C) samples are shown. Melting curves generated by the different ddNTPs are labeled in each panel. Inset: wild-type sample with a 35-mer SNE oligonucleotide. ddATP = thin black solid line, ddTTP = thin gray dashed line, ddCTP = thin black dashed line, ddGTP = thick gray solid line, and no extension control = thick black solid line. The Journal of Molecular Diagnostics 2010 12, 731-738DOI: (10.2353/jmoldx.2010.090149) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 5 SNE of F2 g.21538G>A (A), F5 c.1601G>A (B), MTHFR c.665C>T (C), and MTHFR c.1286A>C (D). Each target is labeled with the ddNTP that was used to genotype the SNP. The different genotypes are labeled in each panel. For each target, the wild-type genotype = thick black line, heterozygous genotype = thin black line, and the homozygous mutant genotype = thick gray line. The Journal of Molecular Diagnostics 2010 12, 731-738DOI: (10.2353/jmoldx.2010.090149) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions