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JMD 2005, Vol. 7, No. 3
Copyright © 2005 American Society for Investigative Pathology & Association for Molecular Pathology

Sensitive Sequencing Method for KRAS Mutation Detection by Pyrosequencing

Shuji Ogino*{dagger}{ddagger}, Takako Kawasaki{dagger}, Mohan Brahmandam{dagger}, Liying Yan§, Mami Cantor{dagger}, Chungdak Namgyal{dagger}, Mari Mino-Kenudson{ddagger}, Gregory Y. Lauwers{ddagger}, Massimo Loda*{dagger}{ddagger} and Charles S. Fuchs||{dagger}{ddagger}

From the Departments of Pathology * and Medicine, || Brigham and Women’s Hospital, Boston; the Department of Medical Oncology, {dagger} Dana-Farber Cancer Institute, Boston; Harvard Medical School, {ddagger} Boston; Biotage AB and Biosystems, § Foxborough; and the Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts

Both benign and malignant tumors represent heterogenous tissue containing tumor cells and non-neoplastic mesenchymal and inflammatory cells. To detect a minority of mutant KRAS alleles among abundant wild-type alleles, we developed a sensitive DNA sequencing assay using Pyrosequencing, ie, nucleotide extension sequencing with an allele quantification capability. We designed our Pyrosequencing assay for use with whole-genome-amplified DNA from paraffin-embedded tissue. Assessing various mixtures of DNA from mutant KRAS cell lines and DNA from a wild-type KRAS cell line, we found that mutation detection rates for Pyrosequencing were superior to dideoxy sequencing. In addition, Pyrosequencing proved superior to dideoxy sequencing in the detection of KRAS mutations from DNA mixtures of paraffin-embedded colon cancer and normal tissue as well as from paraffin-embedded pancreatic cancers. Quantification of mutant alleles by Pyrosequencing was precise and useful for assay validation, monitoring, and quality assurance. Our Pyrosequencing method is simple, robust, and sensitive, with a detection limit of approximately 5% mutant alleles. It is particularly useful for tumors containing abundant non-neoplastic cells. In addition, the applicability of this assay for DNA amplified by whole-genome amplification technique provides an expanded source of DNA for large-scale studies.




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