This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Liu, W.-H. Articles by Makrigiorgos, G. M. Search for Related Content PubMed PubMed Citation Articles by Liu, W.-H. Articles by Makrigiorgos, G. M.

Inverse PCR-Based RFLP Scanning Identifies Low-Level Mutation Signatures in Colon Cells and Tumors

Department of Radiation Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts

Detecting the presence and diversity of low-level mutations in human tumors undergoing genomic instability is desirable due to their potential prognostic value and their putative influence on the ability of tumors to resist drug treatment and/or metastasize. However, direct measurement of these genetic alterations in surgical samples has been elusive, because technical hurdles make mutation discovery impractical at low-mutation frequency levels (<10^–2). Here, we describe inverse PCR-based amplified restriction fragment length polymorphism (iFLP), a new technology that combines inverse PCR, RFLP, and denaturing high-performance liquid chromatography to allow scanning of the genome at several thousand positions per experiment for low-level point mutations. Using iFLP, widespread, low-level mutations at mutation frequency 10^–2–10^–4 were discovered in genes located on different chromosomes, e.g., OGG1, MSH2, PTEN, ß-catenin, Bcl-2, P21, ATK3, and Braf, in human colon cancer cells that harbor mismatch repair deficiency whereas mismatch repair-proficient cells were mutation free. Application of iFLP to the screening of sporadic colon cancer surgical specimens demonstrated widespread low-level mutations in seven out of 10 samples, but not in their normal tissue counterparts, and predicted the presence of millions of diverse, low-incidence mutations in tumors. Unique low-level mutational signatures were identified for each colon cancer cell line and tumor specimen. iFLP allows the high-throughput discovery and tracing of mutational signatures in human cells, precancerous lesions, and primary or metastatic tumors and the assessment of the number and heterogeneity of low-level mutations in surgical samples.

This article has been cited by other articles:

				C. A. Milbury, J. Li, and G. M. Makrigiorgos PCR-Based Methods for the Enrichment of Minority Alleles and Mutations Clin. Chem., April 1, 2009; 55(4): 632 - 640. [Abstract] [Full Text] [PDF]

				G. Wang, E. Maher, C. Brennan, L. Chin, C. Leo, M. Kaur, P. Zhu, M. Rook, J. L. Wolfe, and G. M. Makrigiorgos DNA amplification method tolerant to sample degradation Genome Res., November 1, 2004; 14(11): 2357 - 2366. [Abstract] [Full Text] [PDF]