Published online 28 October 2004
Nucleic Acids Research, Vol. 32 No. 19 © Oxford University Press 2004; all rights reserved
High sensitivity EndoV mutation scanning through real-time ligase proofreading
Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, NY 10021, USA, 1 Applied Biosystems, Foster City, CA 94404, USA, 2 Colorectal Service, Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY 10021, USA and 3 Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA
* To whom correspondence should be addressed. Tel: +1 212 746 6509; Fax: +1 212 746 7983; Email: barany{at}med.cornell.edu
Received September 9, 2004; Revised and Accepted October 11, 2004
The ability to associate mutations in cancer genes with the disease and its subtypes is critical for understanding oncogenesis and identifying biomarkers for clinical diagnosis. A two-step mutation scanning method that sequentially used endonuclease V (EndoV) to nick at mismatches and DNA ligase to reseal incorrectly or nonspecifically nicked sites was previously developed in our laboratory. Herein we report an optimized single-step assay that enables ligase to proofread EndoV cleavage in real-time under a compromise between buffer conditions. Real-time proofreading results in a dramatic reduction of background cleavage. A universal PCR strategy that employs both unlabeled gene-specific primers and labeled universal primers, allows for multiplexed gene amplification and precludes amplification of primer dimers. Internally labeled PCR primers eliminate EndoV cleavage at the 5' terminus, enabling high-throughput capillary electrophoresis readout. Furthermore, signal intensity is increased and artifacts are reduced by generating heteroduplexes containing only one of the two possible mismatches (e.g. either A/C or G/T). The single-step assay improves sensitivity to 1:50 and 1:100 (mutant:wild type) for unknown mutations in the p53 and K-ras genes, respectively, opening prospects as an early detection tool.
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