SurveyorTM nuclease-based detection of p53 gene mutations in haematological malignancy

Abstract

Background: SurveyorTM nuclease is a new single-strand-specific endonuclease that cleaves heteroduplex DNA at a base-mismatch site in both DNA strands. We applied this enzyme to detection of p53 gene mutations in haematological malignancy. Methods: DNA fragments including exons 5 and 6, and those including exons 7 and 8, of the p53 gene, were amplified by polymerase chain reaction using DNA samples extracted from bone marrow aspirates. Denaturation followed by gradual annealing of the amplified fragments formed a heteroduplex with a base-mismatch if a mutation existed because the DNA samples contained wild-type DNA derived from coexisting non-malignant cells. After cleavage by Surveyor nuclease, mutations were simply detected by gel electrophoresis as extra bands of shorter size. Results: Somatic mutations were clearly detected by this method in three of 39 different samples and confirmed by sequencing. The limit of detection estimated by changing the proportion of heteroduplexes in hetero/homoduplexes was between 1/8 and 1/16. Conclusion: We suggest that our method is not only simple, but also sensitive, compared with other complicated methods, and would therefore be useful in current clinical laboratory settings.