The utility of optical waveguide DNA array hybridization and melting for rapid resolution of mismatches, and for detection of minor mutant components in the presence of a majority of wild type sequence: Statistical model and supporting data.
We have derived a statistical model to determine binding and dissociation kinetics of oligonucleotide-antibody-microparticle complexes to oligonucleotides on a surface illuminated by the evanescent wave. The model supports the extremely rapid binding kinetics previously observed and predicts very rapid thermal dissociation kinetics. Experimental results confirmed the model and showed that the most rapid heating available achieved meltoff in 8 s with discrimination of single base differences. Similarly, a model of particulate labels with multiple points of interaction predicted that the system would be very sensitive to a minority of matched interactions in the presence of a majority of mismatched interactions. Conversely, the system would be relatively insensitive to the presence of a higher proportion of matched species. These predictions are born out by experiment. A 1:10 mixture of matched:mismatched gave a melting curve which was approximately the sum of melting curves of the two species alone, and a 50:50 mixture gave a melting curve almost indistinguishable from the highest melting component. Thus, the system will be amenable to detection of infrequent mutations in the presence of an excess of the wild type allele provided the mutant sequence is present on the chip. Further, it lends itself to high throughput automation due to the rapidity with which the thermal melt can be achieved.
Stimpson
D I
DI
Haviland Meteorite Crater, KS 67059, USA. 750-3797@mcimail.com
Gordon
J
J
D004247:DNA / Q000032:analysis*; D007700:Kinetics; D015233:Models, Statistical; D009693:Nucleic Acid Hybridization; D055095:Optics and Photonics
D004247:DNA