High sensitivity EndoV mutation scanning through real-time ligase proofreading. Academic Article uri icon

Overview

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.

publication date

  • October 28, 2004

Research

keywords

  • DNA Ligases
  • DNA Mutational Analysis
  • Deoxyribonuclease (Pyrimidine Dimer)
  • Neoplasms
  • Polymerase Chain Reaction

Identity

PubMed Central ID

  • PMC528826

Scopus Document Identifier

  • 16544377178

Digital Object Identifier (DOI)

  • 10.1093/nar/gnh150

PubMed ID

  • 15514109

Additional Document Info

volume

  • 32

issue

  • 19