Surveyor nuclease-based detection of p53 gene mutations in haematological malignancy.
BACKGROUND
Surveyor 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.


CONCLUSIONS
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.
Mitani
Noriaki
N
Central Clinical Laboratory, Nara Medical University Hospital, Shijo-Cho 840, Kashihara, Nara 634-8522, Japan.
Niwa
Yoshimasa
Y
Okamoto
Yasuyuki
Y
D020137:Base Pair Mismatch; D004252:DNA Mutational Analysis / Q000379:methods*; D003850:Deoxyribonuclease I / Q000378:metabolism*; D016158:Genes, p53; D019337:Hematologic Neoplasms / Q000235:genetics*; D006801:Humans; D020125:Mutation, Missense; D020641:Polymorphism, Single Nucleotide
D003850:Deoxyribonuclease I