Surveyor nuclease assay

[1] The enzyme recognizes all base substitutions and insertions/deletions, and cleaves the 3′ side of mismatched sites in both DNA strands with high specificity[2] This assay has been used to identify and analyze mutations in a variety of organisms and cell types, as well as to confirm genome modifications following genome editing (using CRISPR/TALENs/zinc fingers).

The ability to discover and detect known and unknown mutations is of great importance in biomedical research and genetic diagnosis (see Applications).

Other widely used methods depend on physical properties of DNA, for example melting temperature-based systems such as Single-stranded conformational polymorphism analysis (SSCP) and Denaturing high-performance liquid chromatography (DHPLC).

These methods are simple to run using standard laboratory techniques and equipment, and can detect polymorphisms, single base pair mismatches, and insertions and deletions at low frequencies.

[3] One of the commonly used enzymes is Surveyor nuclease (CEL II), which cleaves the 3′ side of both DNA strands with high specificity at sites of base substitution or insertion/deletion.

Olekowski and colleagues demonstrated this technique by using this enzyme to detect a variety of mutations and polymorphisms in the human BRCA1 gene.

In 2004, Qiu et al. developed a mutation detection technology based on CEL II, also known as Surveyor Nuclease.

Surveyor nuclease was licensed from the Fox Chase Cancer Center by Transgenomic, Inc. and was subsequently sold to IDT, which currently distributes it.

The region of interest in both mutant and wild-type reference DNA is amplified by polymerase chain reaction (PCR).

The DNA of interest is denatured and annealed in order to form heteroduplexes containing a mismatch at the point of the mutation, which can then be identified by the Surveyor nuclease.

If fluorescent labelled primers are used to mark the 5’ and 3’ end of the PCR products, different colored bands will be observed in the analysis.

[7] Surveyor nuclease is a reasonably sensitive enzyme, producing detectable cleavage products from sequences representing only a small proportion of DNA in the population.

[8] In order to detect multiple mutations in the same fragment, post-PCR clean-up must be done before Surveyor nuclease digestion.

Surveyor nuclease also has a 5′ exonuclease activity that attacks the ends of double‐stranded DNA increasing background signal during extended incubation.

[9][10][11] Even after clonal expansion, detection of mutations using Sanger sequence may be difficult as each allele can undergo a different editing event.

In this case, the Surveyor nuclease assay will actually use this effect to create the required heteroduplexes for detection by the mismatch endonuclease.