Detection of single nucleotide variations by a hybridization proximity assay based on molecular beacons and luminescence resonance energy transfer.
A powerful combination of molecular beacon and luminescence resonance energy transfer technology reveals alterations in nucleic acid structure by as little as a single nucleotide in a novel hybridization proximity assay. The assay measures the length of a single-stranded target when a terbium chelate-labeled molecular beacon hybridizes to one side of the nucleic acid segment to be measured and an acceptor probe carrying a convention fluorophore hybridizes to the opposite end of the target. Using a test sequence shortened incrementally by deleting single nucleotides, this assay reports a nearly linear relationship between sequence length and the distance separating acceptor and donor probes. Consequently, this assay can be used to detect alternative splicing, allele types, rearrangements, insertion, and deletion events by measuring separation distances within a predefined region. Furthermore, the use of terbium chelates in molecular beacons can produce exceptionally high signal-to-background ratios compared to the use of conventional fluorophores. Principles of optimal probe design are investigated experimentally and by computational simulations of plausible molecular beacon folding. Some molecular beacon designs form dimers that reduce their maximal response to target sequences. A simple assay to detect such dimers is reported as a tool to help improve the design of molecular beacons. Optimally designed molecular beacons with terbium chelates and hybridization proximity assays are expected to expand their applications in the analysis and screening of genetic diseases.
Root
Douglas D
DD
Department of Biological Sciences, University of North Texas, Denton, TX 76203, USA. droot@unt.edu
Vaccaro
Carlos
C
Zhang
Zhiling
Z
Castro
Miguel
M
D001483:Base Sequence; D002614:Chelating Agents; D014644:Genetic Variation; D028581:Lanthanoid Series Elements; D008163:Luminescent Measurements; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization / Q000379:methods; D009838:Oligodeoxyribonucleotides; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*
D002614:Chelating Agents; D028581:Lanthanoid Series Elements; D009838:Oligodeoxyribonucleotides