Allele-Specific, Non-Extendable Primer Blocker PCR (AS-NEPB-PCR) for DNA Mutation Detection in Cancer Haiying Wang, John Jiang, Bianca Mostert, Anieta.

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Allele-Specific, Non-Extendable Primer Blocker PCR (AS-NEPB-PCR) for DNA Mutation Detection in Cancer Haiying Wang, John Jiang, Bianca Mostert, Anieta Sieuwerts, John W.M. Martens, Stefan Sleijfer, John A. Foekens, Yixin Wang The Journal of Molecular Diagnostics Volume 15, Issue 1, Pages 62-69 (January 2013) DOI: 10.1016/j.jmoldx.2012.08.007 Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Diagram for NEPB design. Primer with 3′ end modifications (eg, phosphate, inverted dT, or amino-C7) functions as a blocking group to prevent DNA polymerase extension on wild-type (WT) sequence. Real-time AS-PCR amplifies only allele-specific (AS) primed mutation, whereas WT is blocked by the modified non-extendable primer blocker (NEPB). The Journal of Molecular Diagnostics 2013 15, 62-69DOI: (10.1016/j.jmoldx.2012.08.007) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 PCR product detected by gel electrophoresis: equal amounts of PCR products were loaded on the gel. Single sharp bands were observed from 5% to 0.1% mutant-containing reactions, and no PCR products were observed from the wild-type–only reactions. Actin was used as the control. A: B-Raf assay. Lane 1: 100-bp Molecular weight marker. Lanes 2–7: NEPB: HT29 cell line 5%, 1%, 0.5%, 0.1%, SKBR3, and no template control (NTC). Lanes 8–12: Actin control: HT29_1%, 0.5%, 0.1%, SKBR3 and NTC. B: K-Ras P4 (G12V; G>T) assay. Lane 1: 100-bp Molecular weight marker. Lanes 2–4: No blocker: SW480 cell line 0.1%, wild-type (WT) and NTC. Lanes 5–7: NEPB: SW480 0.1%, WT, and NTC. Lanes 8–10: BP: SW480 0.1%, WT, and NTC. Lanes 11 and 12: Actin control: SW480 0.1% and WT. C: K-Ras P7 (G13D; 13G>A) assay. Lane 1: 100-bp Molecular weight marker. Lanes 2–4: No blocker: HCT116 cell line 0.1%, WT, and NTC; Lanes 5–7: NEPB: HCT116 0.1%, WT and NTC; Lanes 8–10. BP: HCT116 0.1%, WT, and NTC. Lanes 11 and 12: Actin control: HCT116 0.1% and WT. D: EGFR (L858R; 2573 T>G) assay. Lane 1: 100-bp Molecular weight marker. Lanes 2–5: No blocker: H1975 cell line 1%, 0.5%, 0.1%, and NCI-H358 cell line (WT). Lanes 6–9: NEPB: H1975 1%, 0.5%, 0.1%, and NCI-H358 (WT). Lanes 10–12: Actin control: NCI-H358 (WT) and NTC. The Journal of Molecular Diagnostics 2013 15, 62-69DOI: (10.1016/j.jmoldx.2012.08.007) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions