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  • Original Paper
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Multiplex PCR/LDR for detection of K-ras mutations in primary colon tumors

Oncogene volume 18, pages 27–38 (1999)Cite this article

Abstract

Point mutations in codons 12, 13, and 61 of the K-ras gene occur early in the development of colorectal cancer and are preserved throughout the course of tumor progression. These mutations can serve as biomarkers for shed or circulating tumor cells and may be useful for diagnosis of early, curable tumors and for staging of advanced cancers. We have developed a multiplex polymerase chain reaction/ligase detection reaction (PCR/LDR) method which identifies all 19 possible single-base mutations in K-ras codons 12, 13, and 61, with a sensitivity of 1 in 500 wild-type sequences. In a blinded study, 144 paraffin-embedded archival colon carcinomas were microdissected and K-ras mutations determined by both dideoxy-sequencing and multiplex PCR/LDR. Results were concordant for 134 samples. The ten discordant samples were re-evaluated using higher sensitivity uniplex PCR/LDR, and the original multiplex PCR/LDR result was confirmed in nine of these ten cases. Multiplex PCR/LDR was able to identify mutations in solid tumors or paraffin-embedded tissues containing a majority of wild-type stromal cells, with or without microdissection. The technique is well suited for large scale studies and for analysis of clinical samples containing a minority population of mutated cells.

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Acknowledgements

We thank Michael Wigler, Jerard Hurwitz, Michael Osborne, Alfred Cohen and Daniel Knowles for helpful discussions. We thank the other members of the Barany and Paty laboratories for technical assistance and helpful discussion. MZ is supported by an NIH Medical Scientist Training Program Fellowship at the Tri-institutional MD/PhD program. This work was supported by Grants from the National Cancer Institute (CA65930-02) and Applied Biosystems Division of Perkin-Elmer Corporation.

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Authors and Affiliations

  1. Department of Microbiology, Box 62, Hearst Microbiology Research Center, Strang Cancer Prevention Center, Joan and Sanford I Weill Medical College of Cornell University, 1300 York Avenue, New York, 10021, NY, USA

    Marilyn Khanna, Monib Zirvi, Weiguo Cao, Joseph Day & Francis Barany

  2. Department of Surgery, Colorectal service, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, Room C-983, New York, 10021, NY, USA

    Pat Park, Antonio Picon & Philip Paty

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  1. Marilyn Khanna
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Khanna, M., Park, P., Zirvi, M. et al. Multiplex PCR/LDR for detection of K-ras mutations in primary colon tumors. Oncogene 18, 27–38 (1999). https://doi.org/10.1038/sj.onc.1202291

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