Detection of single nucleotide polymorphisms using a DNA Holliday junction nanoswitch--a high-throughput fluorescence lifetime assay.
We report a simple DNA sensor device, using a combination of binding and conformational switching, capable of rapid detection of specific single nucleotide polymorphisms in an unlabelled nucleic acid target sequence. The detection is demonstrated using fluorescence lifetime measurements in a high-throughput micro plate reader instrument based on the time-correlated single-photon counting technique. The sensor design and instrumental architecture are capable of detecting perturbations in the molecular structure of the probe-target complex (which is similar to that of a Holliday junction), due to a single base pair mismatch in a synthetic target. Structural information, including fluorophore separations, is obtained using time-resolved Förster resonance energy transfer between two fluorophores covalently bound to the probe molecule. The two probes required are designed to detect a single nucleotide polymorphism from a sequence present on each of the two copies of human chromosome 11.
McGuinness
Colin D
CD
School of Physics & Astronomy, James Clerk Maxwell Building, University of Edinburgh, Mayfield Road, Edinburgh, UK. colin.mcguinness@ed.ac.uk
Nishimura
Mira K Y
MK
Keszenman-Pereyra
David
D
Dickinson
Paul
P
Campbell
Colin J
CJ
Bachmann
Till T
TT
Ghazal
Peter
P
Crain
Jason
J
D015374:Biosensing Techniques / Q000379:methods*; D002880:Chromosomes, Human, Pair 11; D045566:DNA, Cruciform / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D036103:Nanotechnology / Q000379:methods*; D009690:Nucleic Acid Conformation; D020641:Polymorphism, Single Nucleotide
D045566:DNA, Cruciform