TY - JOUR
T1 - Detection of tumor mutations in the presence of excess amounts of normal DNA
AU - Sun, Xiyuan
AU - Hung, K.
AU - Wu, L.
AU - Sidransky, D.
AU - Guo, Baochuan
N1 - Funding Information:
Acknowledgments This work was supported by a grant from Variagenics, Inc. M.J.S. is the recipient of fellowships from the Alfred and Isabel Bader Foundation and from Hoffman-La Roche and Eli Lilly. A.E. gratefully acknowledges support from the Swiss National Science Foundation (postdoctoral fellowship 1998–1999) and from Harvard University (1999–2000). We thank Jia Wolfe, Jeff Olson, and Tomohiko Kawate of Variagenics, Inc. for sharing unpublished results, and members of the Verdine group for valuable discussions.
Funding Information:
Acknowledgments This work was supported by NIH grants HG01815 and CA81653 (B.G.).
PY - 2002/2
Y1 - 2002/2
N2 - Mutations are important markers in the early detection of cancer. 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 specific. In addition, they should be possible to carry out in an automated and high-throughput manner to allow large-scale screening. 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.
AB - Mutations are important markers in the early detection of cancer. 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 specific. In addition, they should be possible to carry out in an automated and high-throughput manner to allow large-scale screening. 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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U2 - 10.1038/nbt0202-186
DO - 10.1038/nbt0202-186
M3 - Article
C2 - 11821866
AN - SCOPUS:0036168249
SN - 1087-0156
VL - 20
SP - 186
EP - 189
JO - Nature biotechnology
JF - Nature biotechnology
IS - 2
ER -