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

Irina A. Afonina; Sergey G. Lokhov; Yevheniy S. Belousov; Michael W. Reed; Eugeny A. Lukhtanov; Irina G. Shishkina; Vladimir V. Gorn; Silvia M. Sanders; David K. Walburger; Merl F. Hoekstra; Nicolaas M. J. Vermeulen

doi:10.1117/12.472099

21 June 2002 Hybridization-triggered fluorescence detection of DNA with minor groove binder-conjugated probes

Irina A. Afonina, Sergey G. Lokhov, Yevheniy S. Belousov, Michael W. Reed, Eugeny A. Lukhtanov, Irina G. Shishkina, Vladimir V. Gorn, Silvia M. Sanders, David K. Walburger, Merl F. Hoekstra, Nicolaas M. J. Vermeulen

Author Affiliations +

Proceedings Volume 4626, Biomedical Nanotechnology Architectures and Applications; (2002) https://doi.org/10.1117/12.472099
Event: International Symposium on Biomedical Optics, 2002, San Jose, CA, United States

Abstract

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. 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 order 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, final 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.

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Irina A. Afonina, Sergey G. Lokhov, Yevheniy S. Belousov, Michael W. Reed, Eugeny A. Lukhtanov, Irina G. Shishkina, Vladimir V. Gorn, Silvia M. Sanders, David K. Walburger, Merl F. Hoekstra, and Nicolaas M. J. Vermeulen "Hybridization-triggered fluorescence detection of DNA with minor groove binder-conjugated probes", Proc. SPIE 4626, Biomedical Nanotechnology Architectures and Applications, (21 June 2002); https://doi.org/10.1117/12.472099

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