Introduction to the Workshop
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The electronic form of this document may be cited in the following style:
Human Genome Program, U.S. Department of Energy, DOE Human Genome Program Contractor-Grantee Workshop IV, 1994.
Abstracts scanned from text submitted for November 1994 DOE Human Genome Program Contractor-Grantee Workshop. Inaccuracies have not been corrected.
PHOTO-ANCHORING PROBES FOR IN-SITU LOCALIZATION
A. I. Poletaev*, T. V. Nasedkina#, T. S. Godovikova* and D. G. Knorre*;
#Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 117984, Moscow; *Novosibirsky Institute of Bioorganic Chemistry RAS, 630090, Novosibirsk, Russia.
A new version of DNA probe localization technology on chromosomes and chromatin is suggested. It is based on use of the nucleotide analogs able to cross-link with matrixed DNA in the course of photo-reaction stimulated by light of appropriate wavelength. We used phenilazide active group (4-azidobenzoic, 4-azido-2-nitroaniline and 4-azido-2,3,5,6-tetrafluorobenzoic) linked in 5-position to deoxyuridine (PAdU). After formation of covalent links between the probe and chromosomal DNA, excess probe can be removed from the sample by extensive washing at high temperature (up to 70°C), thus providing a high contrast and a possibility of conducting the second hybridization. Two versions of this technology were proven: with polymerase reaction (Klenow fragment) primed by hybridized DNA probes of different length and conducting with photo-active analogs of dNTP; and by direct incorporation of photo-active nucleotides in the probes by nick-translation using two types of modified dNTP (photoactive and fluorescent). In the first case only the perfect duplexes formed by DNA probes with matrice supply the polymerase extension of the probes and become anchored to matrice after irradiation, that is more suitable for localization of small probes. Extensive washing removes most of the non-specific signals from the samples. The second approach is more simple for bigger probes. Both technologies will be illustrated by examples. The further development of this approach might provide a possibility of localization of very short probes both on chromosomes and on interphase chromatin. This work was supported by a grant from Russian State Program "Human Genome".