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Detection of tumor mutations in the presence of excess amounts of normal DNA

Nature Biotechnology volume 20, pages 186189 (2002) | Download Citation



Mutations are important markers in the early detection of cancer1,2,3. Clinical specimens such as bodily fluid samples often contain a small percentage of mutated cells in a large background of normal cells. Thus, assays to detect mutations leading to cancer need to be highly sensitive and specific4,5,6,7,8. In addition, they should be possible to carry out in an automated and high-throughput manner to allow large-scale screening4,5,6,7,8. Here we describe a screening method, termed PPEM (PNA-directed PCR, primer extension, MALDI-TOF), that addresses these needs more effectively than do existing methods. DNA samples are first amplified using peptide nucleic acid (PNA)–directed PCR clamping reactions in which mutated DNA is preferentially enriched. The PCR-amplified DNA fragments are then sequenced through primer extension to generate diagnostic products. Finally, mutations are identified using matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. This method can detect as few as 3 copies of mutant alleles in the presence of a 10,000-fold excess of normal alleles in a robust and specific manner. In addition, the method can be adapted for simultaneous detection of multiple mutations and is amenable to high-throughput automation.

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This work was supported by NIH grants HG01815 and CA81653 (B.G.).

Author information


  1. Department of Chemistry, Cleveland State University, Cleveland, OH 44115.

    • Xiyuan Sun
    • , K. Hung
    •  & Baochuan Guo
  2. Department of Otolaryngology–Head and Neck Surgery, Johns Hopkins School of Medicine, Baltimore, MD 21287.

    • L. Wu
    •  & D. Sidransky


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Correspondence to Baochuan Guo.

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