Use of extremely short Förster resonance energy transfer probes in real-time polymerase chain reaction.

Described in the article is a new approach for the sequence-specific detection of nucleic acids in real-time polymerase chain reaction (PCR) using fluorescently labeled oligonucleotide probes. The method is based on the production of PCR amplicons, which fold into dumbbell-like secondary structures carrying a specially designed 'probe-luring' sequence at their 5' ends. Hybridization of this sequence to a complementary 'anchoring' tail introduced at the 3' end of a fluorescent probe enables the probe to bind to its target during PCR, and the subsequent probe cleavage results in the florescence signal.

Use of base modifications in primers and amplicons to improve nucleic acids detection in the real-time snake polymerase chain reaction.

The addition of relatively short flap sequence at the 5'-end of one of the polymerase chain reaction (PCR) primers considerably improves performance of real-time assays based on 5'-nuclease activity. This new technology, called Snake, was shown to supersede the conventional methods like TaqMan, Molecular Beacons, and Scorpions in the signal productivity and discrimination of target polymorphic variations as small as single nucleotides. The present article describes a number of reaction conditions and methods that allow further improvement of the assay performance.

New approach to real-time nucleic acids detection: folding polymerase chain reaction amplicons into a secondary structure to improve cleavage of Forster resonance energy transfer probes in 5'-nuclease assays.

The article describes a new technology for real-time polymerase chain reaction (PCR) detection of nucleic acids. Similar to Taqman, this new method, named Snake, utilizes the 5'-nuclease activity of Thermus aquaticus (Taq) DNA polymerase that cleaves dual-labeled Förster resonance energy transfer (FRET) probes and generates a fluorescent signal during PCR. However, the mechanism of the probe cleavage in Snake is different.

Use of base-modified duplex-stabilizing deoxynucleoside 5'-triphosphates to enhance the hybridization properties of primers and probes in detection polymerase chain reaction.

Several base-modified duplex-stabilizing deoxyribonucleoside 5'-triphosphates (dNTPs) have been evaluated as agents for enhancing the hybridization properties of primers and probes in real-time polymerase chain reaction (PCR). It was shown that pyrimidines substituted at the 5-position with bromine or iodine atoms and methyl or propynyl groups are incorporated into PCR amplicons by Taq DNA polymerase as efficiently as natural dNTPs. The dNTP of 2-aminoadenosine was incorporated somewhat less efficiently than dATP but still supported PCR.

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.

Reduced aggregation and improved specificity of G-rich oligodeoxyribonucleotides containing pyrazolo[3,4-d]pyrimidine guanine bases.

Guanine (G)-rich oligodeoxyribonucleotides (ODNs) can form undesired complexes by self association through non-Watson-Crick interactions. These aggregates can compromise performance of DNA probes and make genetic analysis unpredictable. We found that the 8-aza-7-deazaguanine (PPG), a pyrazolo[3,4-d]pyrimidine analog, reduces guanine self association of G-rich ODNs.