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An endonuclease/ligase based mutation scanning method especially suited for analysis of neoplastic tissue

Oncogene volume 21, pages 1909–1921 (2002)Cite this article

Abstract

Knowledge of inherited and sporadic mutations in known and candidate cancer genes may influence clinical decisions. We have developed a mutation scanning method that combines thermostable EndonucleaseV (Endo V) and DNA ligase. Variant and wild-type PCR amplicons are generated using fluorescently labeled primers, and heteroduplexed. Thermotoga maritima (Tma) EndoV recognizes and primarily cleaves heteroduplex DNA one base 3′ to the mismatch, as well as nicking matched DNA at low levels. Thermus species (Tsp.) AK16D DNA ligase reseals the background nicks to create a highly sensitive and specific assay. The fragment mobility on a DNA sequencing gel reveals the approximate position of the mutation. This method identified 31/35 and 8/8 unique point mutations and insertions/deletions, respectively, in the p53, VHL, K-ras, APC, BRCA1, and BRCA2 genes. The method has the sensitivity to detect K-ras mutations diluted 1 : 20 with wild-type DNA, a p53 mutation in a 1.7 kb amplicon, and unknown p53 mutations in pooled DNA samples. EndoV/Ligase mutation scanning combined with PCR/LDR/Universal array proved superior to automated DNA sequencing for detecting p53 mutations in colon tumors. This technique is well suited for scanning low-frequency mutations in pooled samples and for analysing tumor DNA containing a minority of the unknown mutation.

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Acknowledgements

We thank Berton Zbar, Alan Friedman, Donald Bergstrom, Arnold Levine, David Thaler, Richard Cunningham, Daniel Notterman, Ken Offit and Alvaro Monteiro for helpful discussion and Norman Gerry, Carrie Shawber, Jing Lu, Jie Tong and Andrew Grace for their suggestions and technical assistance. We thank Yoke Kow for providing E.coli Endo V and helpful suggestions. We thank Laura Schmidt of the National Cancer Institute-Frederick Cancer Center for providing germline samples containing VHL mutations. We thank Harry Ostrer and Carol Oddoux from the Human Genetics Program at New York University Medical College for providing blinded genomic DNA samples from Ashkenazi Jewish individuals previously typed for the APCI1307K allele. We thank Steven Narod of the University of Toronto for providing samples containing the Ashkenazi Jewish BRCA1 and BRCA2 founder mutations. This work was supported by grants from the National Cancer Institute (P01-CA65930 and RO1-CA81467).

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  1. Weiguo Cao

    Present address: Department of Genetics and Biochemistry, South Carolina Experiment Station, Clemson University, 122 Long Hall, Clemson, 29634-0324, SC, USA

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, Room B-406, 1300 York Avenue, New York, 10021, NY, USA

    Jianmin Huang, Brian Kirk, Reyna Favis, Weiguo Cao & Francis Barany

  2. Laboratoire de Génotoxicologie des tumeurs, Institut Curie, 26 rue d'Ulm, Paris, 75248, France

    Thierry Soussi

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

    Philip Paty

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Huang, J., Kirk, B., Favis, R. et al. An endonuclease/ligase based mutation scanning method especially suited for analysis of neoplastic tissue. Oncogene 21, 1909–1921 (2002). https://doi.org/10.1038/sj.onc.1205109

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