Rapid Screening Assay for KRAS Mutations by the Modified Smart Amplification Process Kenji Tatsumi, Yasumasa Mitani, Jun Watanabe, Hideki Takakura, Kanako.

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Rapid Screening Assay for KRAS Mutations by the Modified Smart Amplification Process Kenji Tatsumi, Yasumasa Mitani, Jun Watanabe, Hideki Takakura, Kanako Hoshi, Yuki Kawai, Takeshi Kikuchi, Yasushi Kogo, Atsuko Oguchi-Katayama, Yasuhiro Tomaru, Hajime Kanamori, Masaru Baba, Takefumi Ishidao, Kengo Usui, Masayoshi Itoh, Paul E. Cizdziel, Alexander Lezhava, Michio Ueda, Yasushi Ichikawa, Itaru Endo, Shinji Togo, Hiroshi Shimada, Yoshihide Hayashizaki The Journal of Molecular Diagnostics Volume 10, Issue 6, Pages 520-526 (November 2008) DOI: 10.2353/jmoldx.2008.080024 Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Schematic of PNA-clamp SMAP-2 assay. For this assay, the SMAP-2 primer design principle is engineered to allow amplification of all possible sequences dictated by the first two nucleotides in codon 12 of KRAS. PNA-clamp competitive probe is designed for the wild-type allele sequence. The greater stability of the PNA probe in hybridization inhibiting to SMAP-2 amplification, and consequently the wild-type allele amplification is suppressed. This effect is theoretically manifest at the initial elongating event from the turn-back primer on wild-type template (case 1) or on a folding primer-synthesized strand (case 2) also early in the reaction. The Journal of Molecular Diagnostics 2008 10, 520-526DOI: (10.2353/jmoldx.2008.080024) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 PNA-clamp SMAP-2 primer set sequences for the KRAS mutation codon 12 detection assay. A: Sequence alignment of the KRAS mutation at codon 12 and SMAP-2 primer positioning. Wild-type sequence of KRAS gene is illustrated. The arrowheads indicate the position of the mutations found in codon 12. DNA sequences used for primer design are marked. B: SMAP-2 primer sequences for the KRAS mutation at codon 12 detection assay. Unlike other SMAP-2 assays previously reported, none of the sequences are a discrimination probe, since they bracket the actual site of sequence variability. The Journal of Molecular Diagnostics 2008 10, 520-526DOI: (10.2353/jmoldx.2008.080024) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Assay validation for detection of the KRAS point mutations at codon 12. Amplification curves of PNA clamp SMAP-2 assay for KRAS mutation detection indicate a detection time of ∼25–35 minutes on homogeneous cell populations harboring KRAS mutations at codon 12. As expected, the heterozygous cell line (closed squares) (KP-1N; GAT, heterozygous) displays slightly slower amplification, likely because it has 50% less actual target sequences than the homozygous cell line (closed circles) (SW480; GTT, homozygous). No amplification is observed for wild-type DNA (open triangles) or the no template DNA (x) controls. The Journal of Molecular Diagnostics 2008 10, 520-526DOI: (10.2353/jmoldx.2008.080024) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 Assay sensitivity for detection of KRAS point mutations at codon 12 in homogeneous and mixed cell populations. A: Sensitivity of the PNA clamp SMAP-2 assay on a homogeneous target. PNA clamp SMAP-2 assay with serial dilutions of SW480 cell line genomic DNA. Shown are 1000 copies (closed circles), 500 copies (closed squares), 100 copies (open circles), 50 copies (open squares), 10 copies (open triangles), and 0 copy (x). Ten copies of mutant DNA are detected in a 26-μl reaction in ∼45 minutes. B: Sensitivity of the PNA clamp SMAP-2 assay on a heterogeneous target. PNA clamp SMAP-2 assay detects mutations in a mixture of wild-type genomic DNA. Shown are 50% (closed circles), 10% (closed squares), 1% (open circles), 0.5% (open squares), 0.1% (open triangles), and 0% (x) of SW480 cell line genomic DNA. Even 0.1% of mutant DNA can be detected in a 26-μl reaction in ∼50 minutes. The Journal of Molecular Diagnostics 2008 10, 520-526DOI: (10.2353/jmoldx.2008.080024) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 5 KRAS codon 12 mutant allele detection of six known variants. Amplification of six mutant sequences corresponding to known mutations of KRAS codon 12 is shown. Each assay was performed on 6000 copies of template. The assay showed little to no bias, validating its effectiveness as a general screening tool for the six mutations of interest. The Journal of Molecular Diagnostics 2008 10, 520-526DOI: (10.2353/jmoldx.2008.080024) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 6 Comparison of PNA and conventional CP for suppression of background amplification in detection of KRAS mutations at codon 12. Shown are 40 ng of SW480 cell line DNA (GTT, homozygous) as a mutant template (closed circles) and 40 ng of wild-type human genomic DNA (GGT, homozygous) as a wild-type template (open squares). A: SMAP-2 amplification without the CP and PNA shows that amplifications of both wild-type and mutant allele were detected at the same time (∼25 minutes). B: SMAP-2 amplification with a conventional CP cannot suppress wild-type allele amplification completely. In this study, 28 different CP sequence designs were tested. Typical data are shown. C: SMAP-2 amplification with a PNA-clamp competitive probe could suppress wild-type allele background amplification completely. The Journal of Molecular Diagnostics 2008 10, 520-526DOI: (10.2353/jmoldx.2008.080024) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions