Detection of BRCA1 and BRCA2 Ashkenazi Jewish Founder Mutations in Formalin- Fixed Paraffin-Embedded Tissues Using Conventional PCR and Heteroduplex/Amplicon.

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Detection of BRCA1 and BRCA2 Ashkenazi Jewish Founder Mutations in Formalin- Fixed Paraffin-Embedded Tissues Using Conventional PCR and Heteroduplex/Amplicon Size Differences Kathy A. Mangold, Vivien Wang, Scott M. Weissman, Wendy S. Rubinstein, Karen L. Kaul The Journal of Molecular Diagnostics Volume 12, Issue 1, Pages 20-26 (January 2010) DOI: 10.2353/jmoldx.2010.090023 Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Bioanalyzer electrophoresis analysis of conventional PCR products using DNA from cell lines with known mutations. Lanes 1–4: PCR amplicons generated using primers for BRCA1-187delAG (wild-type = 90 bp); lanes 5–8: PCR amplicons generated using primers for BRCA1-5385insC (wild-type = 84 bp); lanes 9–12: PCR amplicons generated using primers for BRCA2-6174delT (wild-type = 97 bp). Water only was added to the reactions for lanes 1, 5, and 9; 100 ng of DNA from cell line GM14090 was added to the reactions for lanes 2, 6, and 10; 100 ng of DNA from cell line GM14091 was added to the reactions for lanes 3, 7, and 11; and 100 ng of DNA from cell line GM14170 was added to the reactions for lanes 4, 8, and 12. Heterozygous specimens are shown in lanes 2, 7, and 12. The Journal of Molecular Diagnostics 2010 12, 20-26DOI: (10.2353/jmoldx.2010.090023) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 Bioanalyzer electrophoresis analysis of conventional PCR products using DNA from FFPE clinical specimens. A: PCR amplicons generated using primers for BRCA1-187delAG (wild-type = 90 bp). B: PCR amplicons generated using primers for BRCA1-5385insC (wild-type = 84 bp). C: PCR amplicons generated using primers for BRCA2-6174delT (wild-type = 97 bp). For each mutation assay, only water was added to the reaction for lane 1, and 100 ng of DNA from the appropriate positive cell line was added to the reaction for lane 2. Lanes 3 and 4: DNA from FFPE specimens with no known BRCA mutation; lane 5: DNA from an FFPE specimen with only the BRCA1-S1130X mutation; lanes 6 and 7: DNA from FFPE specimens with BRCA1-187delAG mutations; lanes 8 and 9: DNA from FFPE specimens with BRCA1-5385insC mutations; lanes 10 and 11: DNA from FFPE specimens with BRCA2-6174delT mutations; lane 12: DNA from an FFPE specimen with both the BRCA1-187delAG and BRCA2-6174delT mutations. Heterozygous specimens are shown in lanes A2, A6, A7, A12, B2, B8, B9, C2, C10, C11 and C12. The Journal of Molecular Diagnostics 2010 12, 20-26DOI: (10.2353/jmoldx.2010.090023) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions