A simple, high sensitivity mutation screening using Ampligase mediated T7 endonuclease I and Surveyor nuclease with microfluidic capillary electrophoresis
- 21 March 2012
- journal article
- research article
- Published by Wiley in Electrophoresis
- Vol. 33 (5), 788-796
- https://doi.org/10.1002/elps.201100460
Abstract
Mutation and polymorphism detection is of increasing importance for a variety of medical applications, including identification of cancer biomarkers and genotyping for inherited genetic disorders. Among various mutation-screening technologies, enzyme mismatch cleavage (EMC) represents a great potential as an ideal scanning method for its simplicity and high efficiency, where the heteroduplex DNAs are recognized and cleaved into DNA fragments by mismatch-recognizing nucleases. Thereby, the enzymatic cleavage activities of the resolving nucleases play a critical role for the EMC sensitivity. In this study, we utilized the unique features of microfluidic capillary electrophoresis and de novo gene synthesis to explore the enzymatic properties of T7 endonuclease I and Surveyor nuclease for EMC. Homoduplex and HE DNAs with specific mismatches at desired positions were synthesized using PCR (polymerase chain reaction) gene synthesis. The effects of nonspecific cleavage, preference of mismatches, exonuclease activity, incubation time, and DNA loading capability were systematically examined. In addition, the utilization of a thermostable DNA ligase for real-time ligase mediation was investigated. Analysis of the experimental results has led to new insights into the enzymatic cleavage activities of T7 endonuclease I and Surveyor nuclease, and aided in optimizing EMC conditions, which enhance the sensitivity and efficiency in screening of unknown DNA variations.Keywords
This publication has 33 references indexed in Scilit:
- Screening for Mutations in Kidney-Related Genes Using SURVEYOR Nuclease for Cleavage at Heteroduplex MismatchesThe Journal of Molecular Diagnostics, 2009
- s-RT-MELT for rapid mutation scanning using enzymatic selection and real time DNA-melting: new potential for multiplex genetic analysisNucleic Acids Research, 2007
- Switching base preferences of mismatch cleavage in endonuclease V: an improved method for scanning point mutationsNucleic Acids Research, 2006
- EGFR Mutations in Lung Cancer: Correlation with Clinical Response to Gefitinib TherapyScience, 2004
- RAF/RAS oncogenes and mismatch-repair statusNature, 2002
- Mutations of the BRAF gene in human cancerNature, 2002
- An endonuclease/ligase based mutation scanning method especially suited for analysis of neoplastic tissueOncogene, 2002
- Enzymatic and chemical cleavage methodsElectrophoresis, 1999
- Mutation in the α-Synuclein Gene Identified in Families with Parkinson's DiseaseScience, 1997
- Detection of 81 of 81 Known Mouse β-Globin Promoter Mutations with T4 Endonuclease VII—The EMC MethodGenomics, 1996