Novel DNA probes with low background and high hybridization-triggered fluorescence.

Novel fluorogenic DNA probes are described. The probes (called Pleiades) have a minor groove binder (MGB) and a fluorophore at the 5'-end and a non-fluorescent quencher at the 3'-end of the DNA sequence. This configuration provides surprisingly low background and high hybridization-triggered fluorescence. Here, we comparatively study the performance of such probes, MGB-Eclipse probes, and molecular beacons. Unlike the other two probe formats, the Pleiades probes have low, temperature-independent background fluorescence and excellent signal-to-background ratios. The probes possess good mismatch discrimination ability and high rates of hybridization. Based on the analysis of fluorescence and absorption spectra we propose a mechanism of action for the Pleiades probes. First, hydrophobic interactions between the quencher and the MGB bring the ends of the probe and, therefore, the fluorophore and the quencher in close proximity. Second, the MGB interacts with the fluorophore and independent of the quencher is able to provide a modest (2-4-fold) quenching effect. Joint action of the MGB and the quencher is the basis for the unique quenching mechanism. The fluorescence is efficiently restored upon binding of the probe to target sequence due to a disruption in the MGB-quencher interaction and concealment of the MGB moiety inside the minor groove.

A novel endonuclease IV post-PCR genotyping system.

Here we describe a novel endonuclease IV (Endo IV) based assay utilizing a substrate that mimics the abasic lesions that normally occur in double-stranded DNA. The three component substrate is characterized by single-stranded DNA target, an oligonucleotide probe, separated from a helper oligonucleotide by a one base gap. The oligonucleotide probe contains a non-fluorescent quencher at the 5' end and fluorophore attached to the 3' end through a special rigid linker. Fluorescence of the oligonucleotide probe is efficiently quenched by the interaction of terminal dye and quencher when not hybridized. Upon hybridization of the oligonucleotide probe and helper probe to their complementary target, the phosphodiester linkage between the rigid linker and the 3' end of the probe is efficiently cleaved, generating a fluorescent signal. In this study, the use of the Endo IV assay as a post-PCR amplification detection system is demonstrated. High sensitivity and specificity are illustrated using single nucleotide polymorphism detection.

Synthesis, characterization, and application of substituted pyrazolopyrimidine nucleosides.

This unit describes, in detail, the preparation of 3-aminopropyl-substituted pyrazolo[3,4-d]pyrimidine analogs of the purines deoxyadenosine (dA) and deoxyguanosine (dG). Phosphoramidite reagents of these so-called aminopropyl-PPA and -PPG nucleosides (AP-PPA and AP-PPG, respectively) allow introduction of amino linkers into internal positions of synthetic DNA strands. Synthesis of suitably protected AP-PPA and AP-PPG phosphoramidites are described. The stepwise alkynylation, hydrogenation, selective protection, and phosphoramidite synthesis is similar for both the PPA and PPG analogs. To demonstrate the application of these reagents, a protocol is given in which a simple DNA strand is synthesized and conjugated to a lipophilic activated ester (dabcyl-SE) to form a stable amide linkage. Utility of this chemistry for preparing internally modified DNA conjugates is discussed.