High sensitivity EndoV mutation scanning through real-time ligase proofreading.
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.
Pincas
Hanna
H
Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, NY 10021, USA.
Pingle
Maneesh R
MR
Huang
Jianmin
J
Lao
Kaiqin
K
Paty
Philip B
PB
Friedman
Alan M
AM
Barany
Francis
F
D016477:Artifacts; D045744:Cell Line, Tumor; D011088:DNA Ligases / Q000378:metabolism*; D004252:DNA Mutational Analysis / Q000379:methods*; D043224:Deoxyribonuclease (Pyrimidine Dimer) / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D009369:Neoplasms / Q000175:diagnosis / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D013997:Time Factors
D005456:Fluorescent Dyes; D043224:Deoxyribonuclease (Pyrimidine Dimer); D011088:DNA Ligases