Fiberoptic DNA sensor array capable of detecting point mutations.

Abstract

The first integrated fiberoptic DNA sensor array capable of simultaneously monitoring multiple hybridization events has been prepared. In this study, 5'-amino-terminal DNA probes were covalently immobilized by reaction with succinimidyl ester residues in acrylamide-based polymer matrices. DNA sensor arrays of three different oligonucleotide probes, p(dA)(18-mer), H-ras wild-type (10-mer), and H-ras mutant (11-mer) were fabricated, and real-time hybridization of 5'-fluorescein isothiocyanate (FITC)-labeled target oligonucleotides to the array was monitored. H-ras wild-type and Eta-ras mutant differ by a one-base substitution (point mutation) and were used to demonstrate the sensor's ability to distinguish point mutations. Thermal studies indicated a 13 degrees C decrease in the Tm of a duplex with a single-base mismatch. The fiberoptic DNA sensor array was used to discriminate a point mutation by monitoring the real-time hybridization of FITC-labeled target oligonucleotides at 54 degrees C and detected labeled-target oligonucleotides in the range 0.2-196 nM. The lower detection limit is approximately an order of magnitude lower than previously reported DNA biosensors. The DNA sensor array was used to positively identify a point mutation of a biotin-primer-labeled (109 bp) PCR product of the H-ras oncogene. The unitary fiberoptic sensor array is highly sensitive, has the ability to determine point mutations, and has the potential to sample submicroliter volumes due to the small volumes of the individual array elements (20 pL).