Target-assembled ExciProbes: application to DNA detection at the level of PCR product and plasmid DNA.
Recently, we introduced a novel exciplex-based approach for detection of nucleic acids using a model DNA-mounted exciplex system, consisting of two 8-mer ExciProbes hybridized to a complementary 16-mer DNA target. We now show, for the first time, that this approach can be used to detect DNA at the level of PCR product and plasmid, when the target sequence (5'-GCCAAACACAGAATCG-3') was embedded in long DNA molecules (PCR products and approximately 3 Kbp plasmid). A remarkably stringent demand is made of the solvent conditions for this exciplex emission to occur, viz., emission is optimal for DNA at 80% trifluoroethanol, even in the plasmid situations, raising the question of the molecular structural basis of this system. We show that a perfectly matched plasmid target can be differentiated from target containing single nucleotide substitutions; hence, ExciProbes could be applied to SNP analysis. The effect of counter cations (Na(+), K(+), and Mg(2+)) and PCR additives on exciplex emission has been also examined.
Walsh
Lindsey
L
Wolfson Centre for Rational Structure-Based Design of Molecular Diagnostics, School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester, M13 9PL, UK.
Gbaj
Abdul
A
Savage
Hannah E
HE
Bacigalupo
M Candelaria Rogert
MC
Bichenkova
Elena V
EV
Douglas
Kenneth T
KT
D001483:Base Sequence; D004247:DNA / Q000032:analysis* / Q000737:chemistry; D015342:DNA Probes / Q000737:chemistry*; D008954:Models, Biological; D008967:Molecular Biology / Q000379:methods; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010957:Plasmids / Q000737:chemistry* / Q000235:genetics; D016133:Polymerase Chain Reaction
D015342:DNA Probes; D004247:DNA