Mutational scanning of PCR products by subtractive oligonucleotide hybridization analysis

Here, we describe a new approach for mutational scanning of PCR products th rough hybridization analysis between complementary oligonucleotides. Sets o f overlapping probe oligonucleotides complementary to wild-type (WT) sequen ce are hybridized to microbead-immobilized PCR products under solution-like conditions. Mismatch-hybridization situations between a mutant sample and probe oligonucleotides result in higher remaining concentrations in solutio n of involved probe oligonucleotides. Post-hybridization supernatants are s ubsequently analyzed for their probe oligonucleotide compositions using sur face plasmon resonance-based biosensor technology Relative remaining probe oligonucleotide concentrations are monitored in real-time through hybridiza tion analysis between probe oligonucleotides and their corresponding sensor -chip immobilized complementary counterparts. This allows for the construct ion of composition diagrams revealing the existence and approximate locatio n of a mutation within an investigated sample DNA sequence. Applied on PCR products derived from clinical samples of microdissected tumor biopsies, si ngle mutations in exons 6 and 7 of the human p53 tumor-suppressor gene were successfully detected and approximately localized.