Enzyme-Based Mutation DetectionDetection of Mutations using SURVEYOR™ NucleaseKnown and unknown mutations can be detected in DNA sequences using SURVEYOR™ Nuclease as outlined in the four-step protocol below. 
"SURVEYOR Nuclease Treatment" is the key step in this protocol and involves the use of SURVEYOR Nuclease, a member of the family of mismatch-specific CEL nucleases derived from celery. SURVEYOR Nuclease recognizes and cleaves all insertions, deletions and base substitutions that may be present in heteroduplexed DNA. In practice, the cleavage reaction is set up by combining the enzyme with the heteroduplexed substrate followed by a 20 minute incubation at 42°C. Cleavage is stopped by adding a Stop Solution.  | Heteroduplexed DNA template | | Mismatch–specific binding of SURVEYOR Nuclease to the heteroduplex | | Cleavage of both strands on the 3’ side of the mismatched base | | Separation of cleavage fragments |
View a schematic presentation of the process: With Sound or Without Sound SURVEYOR Nuclease cleavage products can subsequently be analyzed by sizing them using either a WAVE® System or WAVE® HS System, or standard gel electrophoresis (agarose or polyacrylamide gels), or fluorescent capillary or polyacrylamide gel electrophoresis. Benefits of using SURVEYOR Nuclease for mutation detection- Detection of all types of base-substitution and insertion/deletion mismatches.
- Size of cleavage fragments provides information about the location of the mutation.
- The number of cleavage products indicates whether there are 1 or 2 or 3 or more mutations.
- Mutations can be detected in longer PCR products. 3-kb fragments have been analyzed successfully.
- Pooling capabilities. To improve the throughput, PCR products can be pooled, cleaved with SURVEYOR Nuclease, and analyzed.
- Analysis can be carried out on different platforms.
- All components other than the thermostable DNA polymerase and reagents needed for the PCR amplification step can be provided in a kit for ease of use.
Applications |