) Fluorescent TISH Probes for Cancer-relevant Sequences Gene amplification, substitution and deletion mutations are associated with the molecular pathology of various malignancies, including breast, cervical and gastric cancers, as well as colon and lung cancers and tumors of the nervous system. Multidrug resistance of cancer cells is also associated with gene amplification. Amplification of erbB-2 (HER-2/neu) and N-myc genes is particularly correlated with poor prognosis in breast and cervical cancers and neuroblastoma, respectively. Methods that could simply and reliably detect such aberrations and quantitate them can therefore be of great value for diagnosis, for following the efficacy of treatment, and for reliable prognosis. Currently, detection of point-mutational events represents a major experimental effort. In the case of gene amplification as well, the methods are arduous, require relatively large samples, and they are of variable reliability. This project has a two-fold aim. One is to apply the methodology of TISH (third strand in situ hybridization of fluorescent probes via triplex formation, which avoids the need for DNA denaturation) for the cytogenetic quantitative analysis of these aberrations. TISH has three major advantages: greater sensitivity and quantitative reliability, and importantly, greater sequence specificity. The second parallel aim is to develop suitably intense and non-quenching fluorescent TISH probes. For this purpose, a major effort will be mounted to develop dendritic nuclei with fluors attached by rigid linkers that prohibit their interaction, therefore preventing the quenching of fluorescence. In this way, it is hoped to enhance the sensitivity of fluorescent probe detection by 1-2 orders of magnitude, thereby assuring that TISH probes can be reliably used for analyzing amplified genes and ultimately for detection of mutations in single-copy genes in situ.
| Amosova, Olga; Smith, Alexander; Fresco, Jacques R (2011) The consensus sequence for self-catalyzed site-specific G residue depurination in DNA. J Biol Chem 286:36316-21 |
| Varganov, Y; Amosova, O; Fresco, J R (2007) Third strand-mediated psoralen-induced correction of the sickle cell mutation on a plasmid transfected into COS-7 cells. Gene Ther 14:173-9 |
