High sensitivity EndoV mutation scanning through real-time ligase proofreading.

TitleHigh sensitivity EndoV mutation scanning through real-time ligase proofreading.
Publication TypeJournal Article
Year of Publication2004
AuthorsPincas H, Pingle MR, Huang J, Lao K, Paty PB, Friedman AM, Barany F
JournalNucleic Acids Res
Date Published2004
KeywordsArtifacts, Cell Line, Tumor, Deoxyribonuclease (Pyrimidine Dimer), DNA Ligases, DNA Mutational Analysis, Fluorescent Dyes, Humans, Neoplasms, Polymerase Chain Reaction, Time Factors

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.

Alternate JournalNucleic Acids Res.
PubMed ID15514109
PubMed Central IDPMC528826
Grant ListP01-CA65930 / CA / NCI NIH HHS / United States
R01-CA81467 / CA / NCI NIH HHS / United States

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