Genotyping Single Nucleotide Polymorphisms in Human Genomic DNA with an Automated and Self-Contained PCR Cassette  Dammika P. Manage, Lucy Ma, Jana Lauzon, Anita Howell, Andrew R. Belch, John R. Mackey, Linda M. Pilarski  The Journal of Molecular Diagnostics  Volume 16, Issue 5, Pages 550-557 (September 2014) DOI: 10.1016/j.jmoldx.2014.04.004 Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Expected results for each FGFR2 genotype after cassette PCR with the use of major or minor allele primers. The principle of ASPCR (for rs1219648) is shown. Each primer is designed to match one allele and mismatch the opposite allele. In addition, a mismatch is introduced intentionally to the penultimate base to increase the mismatch.9 DNA amplification progresses where major allele primer matches with the DNA having the homozygous major genotype (A) and where minor allele primer matches with DNA having the homozygous minor genotype (D). DNA amplification does not take place where minor allele primer does not match with the DNA with homozygous major genotype (B) or where major allele primer does not match with the DNA with homozygous minor genotype (C). The Journal of Molecular Diagnostics 2014 16, 550-557DOI: (10.1016/j.jmoldx.2014.04.004) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Preparation of gel capillary cassettes. Glass capillaries with desiccated gel with all of the ingredients needed for PCR except the DNA template, sample (A), empty pan with wax pattern (B), and pan with capillaries laid ready-to-use cassette (C). The Journal of Molecular Diagnostics 2014 16, 550-557DOI: (10.1016/j.jmoldx.2014.04.004) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Melt curve analysis of cassette PCR for each FGFR2 genotype, using major or minor allele primers. A sample with homozygous major for both SNP allele pairs (A), homozygous minor for both SNP allele pairs (B), and heterozygous for both SNP allele pairs (C), and negative control (D). Insets in each figure show the CCD image of the four capillaries tested for both SNPs. Melt peak temperatures vary between 0.2 and 0.5°C. Product identification was confirmed by agarose gel electrophoresis and sequencing. Error bars show the SD from three replicate experiments. M, major allele primers; m, minor allele primers. The Journal of Molecular Diagnostics 2014 16, 550-557DOI: (10.1016/j.jmoldx.2014.04.004) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Buccal swabs are stable for cassette PCR after dry storage at room temperature. MCA curves are shown after amplification of FGFR2 templates in dried swabs stored at room temperature for different intervals of time before processing. Samples with homozygous major (A), homozygous minor (B), or heterozygous genotypes (C). M, major allele; m, minor allele. The Journal of Molecular Diagnostics 2014 16, 550-557DOI: (10.1016/j.jmoldx.2014.04.004) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions