Microscope objective for large-angle fluorescence used for rapid detection of single nucleotide polymorphisms in DNA hybridization.
A new type of microscope objective is used for the rapid detection of sequence-dependent affinity variations in DNA hybridization. We demonstrate that by performing probe/target hybridization on coverslips at room temperature terminal SNPs (single nucleotide polymorphisms) can be detected within seconds. The study of weak pair interaction, such as the association of very short DNA oligomers, requires the use of high analyte concentrations of both partners to generate a detectable amount of associated pairs. The background of high concentrations of unbound fluorescing analyte can easily hide the low signal of a weakly affine reaction and makes association extremely difficult to detect. Fluorescence detection is a powerful approach to analyze minute amounts of material, even single molecules, but it is usually limited to rather low concentrations. This limitation is now overcome due to the new type of microscope objective, which produces an extremely small detection volume at a water/glass interface.
Ruckstuhl
Thomas
T
Physikalisch-Chemisches Institut, Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland. thomas.ruckstuhl@dcu.ie
Krieg
Alexander
A
D002232:Carbocyanines / Q000737:chemistry; D004867:Equipment Design; D017403:In Situ Hybridization / Q000379:methods*; D018613:Microscopy, Confocal / Q000295:instrumentation / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D012680:Sensitivity and Specificity
D002232:Carbocyanines; C085321:cyanine dye 5