Mutational scanning of PCR products by subtractive oligonucleotide hybridization analysis
1999 (English)In: BioTechniques, ISSN 0736-6205, Vol. 26, no 2, 308-+ p.Article in journal (Refereed) Published
Here, we describe a new approach for mutational scanning of PCR products through hybridization analysis between complementary oligonucleotides. Sets of overlapping probe oligonucleotides complementary to wild-type (WT) sequence 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 solution of involved probe oligonucleotides. Post-hybridization supernatants are subsequently analyzed for their probe oligonucleotide compositions using surface plasmon resonance-based biosensor technology Relative remaining probe oligonucleotide concentrations are monitored in real-time through hybridization analysis between probe oligonucleotides and their corresponding sensor-chip immobilized complementary counterparts. This allows for the construction of composition diagrams revealing the existence and approximate location of a mutation within an investigated sample DNA sequence. Applied on PCR products derived from clinical samples of microdissected tumor biopsies, single mutations in exons 6 and 7 of the human p53 tumor-suppressor gene were successfully detected and approximately localized.
Place, publisher, year, edition, pages
1999. Vol. 26, no 2, 308-+ p.
IdentifiersURN: urn:nbn:se:kth:diva-13318ISI: 000078631700024OAI: oai:DiVA.org:kth-13318DiVA: diva2:323696
QC 201006112010-06-112010-06-112010-06-11Bibliographically approved