Open AccessMutational Scanning of PCR Products by Subtractive Oligonucleotide Hybridization Analysis
Author(s) -
Peter Nilsson,
Anita Larsson,
Joakim Lundeberg,
Mathias Uhlén,
PerÅke Nygren
Publication year - 1999
Publication title -
biotechniques/biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/99262rr01
Subject(s) - oligonucleotide , microbiology and biotechnology , oligomer restriction , biology , microbead (research) , nucleic acid thermodynamics , hybridization probe , molecular probe , in situ hybridization , suppression subtractive hybridization , polymerase chain reaction , dna , complementary dna , gene , genetics , base sequence , messenger rna , cdna library
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.