A simple, high sensitivity mutation screening using Ampligase mediated T7 endonuclease I and Surveyor nuclease with microfluidic capillary electrophoresis

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.

Comparison of T7E1 and Surveyor Mismatch Cleavage Assays to Detect Mutations Triggered by Engineered Nucleases

“…This effect has been explained by the combination of the protection provided by the 5′ label against 5′ exonuclease activity, and an increased signal to noise ration since degraded products lose the label (Till et al 2004). The use of a ligase has also been reported to improve the results (Huang et al 2012). However, these ameliorations are associated with a loss of simplicity and an increase in costs.…”

“…Such a nonspecific exonuclease activity can be reduced by the combination of the EMC enzyme with ampligase (Huang et al 2012). In their paper, Huang et al (2012) discuss two important factors affecting the exonuclease activity of the EMC enzymes. The first factor is the buffer used for the enzymatic incubation, and the second factor is the incubation time.…”

T7 Endonuclease I Mediates Error Correction in Artificial Gene Synthesis

“…This endonuclease most possibly recognizes the incorrect impairment of double DNA strands and cleaves DNA near to mismatches in both strands, resulting in the creation of 5 0 -end in DNA fragments. The 5 0 -end exposed phosphate groups are important substrates for 3 0 -5 0 exonuclease digestion [7,15]. The combination of T7 endonuclease I (DNA mismatch cleavage enzyme) with Kod DNA polymerase that has a strong 3 0 -5 0 exonuclease activity, used to generate proofreading activity, strongly contributed to reduce mutations in artificial genes.…”

“…Four of the six endonucleases chosen belong to the P1/S1 nuclease family and display strong primary sequence conservation especially at the active site, where critical catalytic residues are identical between these enzymes. The other two nucleases selected, endonuclease V from E. coli and T7 endonuclease I from bacteriophage T7, were reported as mismatch cleavage enzymes in different studies involving either error removal or mutation detection [7,15]. Genes encoding the six endonucleases were synthesized in vitro with a codon usage optimized for expression in E. coli and cloned into pHTP1 (6HIS tag) expression vector.…”

“…Some of the selected enzymes, such as T7 endonuclease I, display the capacity to cleave mismatch regions in hetero-duplex nucleic acids and have been applied in mutation screening [11,15] and to a lesser degree in repair systems applied in artificial gene synthesis [2,7]. The endonucleases were of prokaryotic or eukaryotic origins (Table 1).…”