ArticleNucleic acids research2004
High sensitivity EndoV mutation scanning through real-time ligase proofreading.
Article in Nucleic acids research, 2004. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 10 papers.
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Who cites it
10 citing papers in PubMed.
- Enzymatic Methods for Mutation Detection in Cancer Samples and Liquid Biopsies.International journal of molecular sciences · 2023Review
- Programmable endonuclease combined with isothermal polymerase amplification to selectively enrich for rare mutant allele fractions.Chinese chemical letters = Zhongguo hua xue kuai bao · 2022Article
- Article
- Endonuclease V: an unusual enzyme for repair of DNA deamination.Cellular and molecular life sciences : CMLS · 2013Review
- Human endonuclease V as a repair enzyme for DNA deamination.Mutation research · 2012Article
- Dissecting endonuclease and exonuclease activities in endonuclease V from Thermotoga maritima.Nucleic acids research · 2011Article
- PCR-based methods for the enrichment of minority alleles and mutations.Clinical chemistry · 2009Review
- The signatures of autozygosity among patients with colorectal cancer.Cancer research · 2008Article
- Switching base preferences of mismatch cleavage in endonuclease V: an improved method for scanning point mutations.Nucleic acids research · 2007Article
- Detection of rare mutant K-ras DNA in a single-tube reaction using peptide nucleic acid as both PCR clamp and sensor probe.Nucleic acids research · 2006Article
Corrections and comments
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Authors and funding
7 authors.
Funding
Abstract
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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Registered trials
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