Detection of DNA point mutation by atom transfer radical polymerization.
We report here a new DNA detection method in which polymer growth in atom transfer radical polymerization (ATRP) is used as a means to amplify detection signals. In this method, DNA hybridization and ligation reactions led to the attachment of ATRP initiators on a solid surface where specific DNA sequences were located. These initiators subsequently triggered the growth of poly(hydroxyethyl methacrylate) (PHEMA) at the end of immobilized DNA molecules and formed polymer brushes. The formation of PHEMA altered substrate opacity, rendering the corresponding spots readily distinguishable to the naked eye. A second ATRP reaction to form branched polymers on the surface drastically improved the visibility of DNA hybridization and significantly shortened the detection time. The resulting polymer film was characterized using infrared spectroscopy, ellipsometry, contact angle measurements, and atomic force microscopy. Direct visualization of 1 fmol of target DNA molecules of interest was demonstrated. A proof-of-principle experiment to detect DNA point mutation was conducted. The perfectly matched DNA targets were distinctively differentiated from those with mutations. The demonstrated capability to detect DNA mutation with direct visualization laid the groundwork for the future development of detector-free testing kits in single-nucleotide polymorphism screenings.
Lou
Xinhui
X
Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695, USA.
Lewis
Matthew S
MS
Gorman
Christopher B
CB
He
Lin
L
D004247:DNA / Q000032:analysis* / Q000235:genetics* / Q000648:ultrastructure; D005609:Free Radicals / Q000737:chemistry; D018625:Microscopy, Atomic Force; D015394:Molecular Structure; D017354:Point Mutation / Q000235:genetics*; D011102:Polyhydroxyethyl Methacrylate / Q000737:chemistry*; D013057:Spectrum Analysis
D005609:Free Radicals; D011102:Polyhydroxyethyl Methacrylate; D004247:DNA