Label-free detection of missense mutations and methylation differences in the p53 gene using optically diffracting hydrogels.
We have developed a novel approach for DNA detection as well as genetic screening of mutations by uniquely combining DNA-responsive and optically diffracting materials. This approach entails the polymerization of a photonic crystal within a hydrogel network that alters the diffraction of light in response to a target DNA strand. The utility of this approach, which permits label-free sensing, was demonstrated via the detection of a target sequence from the DNA binding domain of the major tumor suppressor protein p53. Using a complementary capture probe strand, we were able to detect down to picomole concentrations of the target p53 sequence. Moreover, we demonstrated that this approach could readily detect a single base pair mutation in the target strand, which corresponds to the hotspot cancer mutation R175H in p53. The sensitivity of detection was increased by lowering the rate of annealing of the target strand and adjusting the solution ionic strength during optical characterization. Changes in ionic strength during characterization impact the melting temperature of the bound target DNA and the Donnan potential between the hydrogel and solution, which influence detection. We further showed that this approach is sensitive to epigenetic changes via the detection of a fully methylated form of the target p53 sequence. Ultimately, this approach represents a new paradigm for DNA detection and specifically genetic screening of p53 as well as other disease markers and nucleotide modifications that alter the properties of DNA (e.g., epigenetic alterations and adducts with chemical carcinogens).
Department of Chemical and Biological Engineering, University of Colorado, Boulder, CO 80309, USA. joel.kaar@colorado.edu mark.stoykovich@colorado.edu.
Kelsey I
KI
MacConaghy
Duncan M
DM
Chadly
Mark P
MP
Stoykovich
Joel L
JL
Kaar
D001483:Base Sequence; D015374:Biosensing Techniques / Q000379:methods*; D004247:DNA / Q000737:chemistry / Q000235:genetics; D019175:DNA Methylation; D015342:DNA Probes / Q000737:chemistry / Q000235:genetics; D016158:Genes, p53 / Q000235:genetics*; D020100:Hydrogels; D020125:Mutation, Missense; D009693:Nucleic Acid Hybridization; D055620:Optical Phenomena; D009994:Osmolar Concentration; D044366:Transition Temperature
D015342:DNA Probes; D020100:Hydrogels; D004247:DNA