Book of Abstracts: Albany 2007
June 19-23 2007
Application of Target-assembled Exciplex-based Probes for DNA Detection and Mismatch Discrimination in PCR Products and Plasmid DNA
Detection of specific nucleic acid sequences is commonly achieved by hybridisation of fluorescently labelled oligonucleotide probes to complementary target sequences. Conventional fluorescent labels (e.g., Cy3, Cy5, FAM,) give high background fluorescence, which can result in false positives and problems with assay sensitivity. Fluorescence Resonance Energy Transfer (FRET)-based methods (e.g., Taqman, Molecular Beacons) are subject to inherently poor detection resolution, as FRET operates between 10 and 100 Å, corresponding to 3-30 base pairs.
We have described a new DNA detection method, an exciplex-based split probe approach, which addresses the limitations of background and resolution (1-2). This consists of a target-assembled fluorescent detector, which is split at a molecular level into two signal-silent exci-probes complementary to adjacent regions of the target sequence. The tandem probes are assembled by the target DNA into a 3-dimensional complex (Figure 1). The prototype target sequence, GCCAAACACAGAATCG, which was detected by two 8-mer oligonucleotide probes labelled with 1-pyrenylmethylamine and an N-methyl-N?-naphthalen-1-yl-ethane-1, 2-diamino moiety, via their 5?-and 3?- terminal phosphate groups, respectively. Hybridisation of the tandem 8-mer exciprobes to the 16-mer target (in Tris buffer containing 80% (v/v) trifluoroethanol), followed by excitation at 350 nm resulted in the formation of an excited state complex (exciplex) fluorescing at a much longer wavelength (∼480 nm).
These previous studies using a synthetic oligonucleotide system were limited to hybridisation of 16-mer target to two 8-mer probes. We now describe use of this approach for target sequences embedded in longer DNA constructs. We have demonstrated exciplex detection of target DNA regions at both PCR product and recombinant plasmid level and were also able to differentiate perfectly matched target from target containing single nucleotide substitutions. We also evaluated the importance to exciplex signal intensity of the presence of various counter-cations (Na+, K+, and Mg2+), as well as the effect of common PCR additives (sulfolane, methylsulfone, betaine, and dimethyl sulfoxide).
Figure 1: (a) Exciprobes assembled by their DNA target. A represents a 1-pyrenylmethylamine moiety and B represents an N-methyl-N?-naphthalen-1-yl-ethane-1, 2-diamino moiety. A and B are attached to the oligonucleotides via 5? and 3? terminal phosphate groups, respectively. The DNA target sequence is 5?pdGCCAAACACAGAATCG. The oligonucleotide parts of split-probes 1 and 2 have sequences 5?pdTGTTTGGC and dCGATTCTG3?p, respectively. (b) Shows the structure of the 1-pyrenemethylamine and N?-methyl-N?-naphthalen-1-yl-ethanedi-amine functional groups, R represents an oligonucleotide.
References and Footnotes
Wolfson Centre for Rational Structure-Based Design of Molecular Diagnostics