Another widely used method for screening DNA mismatches, PCR single-stranded conformational polymorphism (SSCP) analysis, has recently been made much more sensitive by using fluorescence detection and capillary electrophoresis for analyses ( 1). However, this approach generates large quantities of unnecessary data. The method of choice for precise mutation and polymorphism analysis has been DNA sequencing. The discovery of unknown mutations and polymorphisms in genome sequences is a vital aspect of widespread research areas, including pharmacogenetics in humans and reverse genetics in model organisms (relating mutant phenotype to genomic DNA sequence information). The entire protocol can be performed in less than a day and is suitable for automated and high-throughput procedures. Unlabeled Surveyor nuclease digestion products can be analyzed using conventional gel electrophoresis or high-performance liquid chromatography (HPLC), while end-labeled digestion products are suitable for analysis by automated gel or capillary electrophoresis.

The technology is highly sensitive, detecting rare mutants present at as low as 1 in 32 copies. Surveyor nuclease technology involves four steps: ( i) PCR to amplify target DNA from both mutant and wild-type reference DNA ( ii) hybridization to form heteroduplexes between mutant and wild-type reference DNA ( iii) treatment of annealed DNA with Surveyor nuclease to cleave heteroduplexes and ( iv) analysis of digested DNA products using the detection/separation platform of choice. Surveyor nuclease cleaves with high specificity at the 3′ side of any mismatch site in both DNA strands, including all base substitutions and insertion/deletions up to at least 12 nucleotides.

This technology is based on a new mismatch-specific DNA endonuclease from celery, Surveyor™ nuclease, which is a member of the CEL nuclease family of plant DNA endonucleases. We have developed a simple and flexible mutation detection technology for the discovery and mapping of both known and unknown mutations.

Mutation Surveyor Demo