Hybridization-triggered fluorescence detection of DNA with minor groove binder-conjugated probes

Fluorogenic 2'-deoxynucleotide probes containing a minor groove binding-quencher compound at the 5'-end and a fluorophore at the 3'-end, were recently described.(1) These probes fluoresce upon hybridization to the complementary target. The 5'-MGB-quencher group prevents 5'-nuclease digestion by Taq polymerase during homogeneous amplification. The 5'-MGB-quencher-oligonucleotide-fluor (MGB-Q-ODN-Fl) probes displayed a dynamic range of 7 orders of magnitude, with an ultimate sensitivity of better than 5 copies per sample. The high sensitivity and specificity is illustrated by the application of the probes in single nucleotide polymorphism detection, viral load determination and gene expression analyses. This paper summarizes new developments in sequence detection, gene expression and SNP analysis using new T-m prediction software to design robust 5'- MGB-Q-ODN-Fl probes. Furthermore, the software is capable of estimating the T-m of probes containing a modified base. Due to G:G self-association, many G-rich probes and primers are poor performers in amplification reactions. The software recognizes such sequences and substitution of G with 6-Amino-1,5-dihydro-pyrazolo[3,4-d]pyrimidin-4-one (PPG) is indicated, when necessary to eliminate G:G self-association. Examples of improved performance of PPG containing primers and probes is demonstrated.