METHOD FOR SYNTHESISING A FLUORESCENTLY LABELLED DNA PROBE FOR A REACTION OF FLUORESCENT IN SITU HYBRIDISATION (FISH)

摘要

FIELD: biotechnology.;SUBSTANCE: invention relates to the field of molecular biology, namely, to methods for synthesising a fluorescent probe intended for detecting deletion of the short arm of chromosome 17 (del 17p). The fluorescently labelled DNA probe for the reaction of fluorescent in situ hybridisation (FISH) is synthesised by using the RP11-89D11 BAC clone to produce a DNA target and integrating a cyanine (Cy3) fluorescent label, followed by stabilisation in a buffer solution with an optimal buffer composition containing: 3 mcl of 10X PCR buffer, 4 mcl of MgCl2 (25 mM), 2 mcl each of DOP primers (15 mcM), 1 mcl each of dATP, dTTP, dGTP (8 mM), 2 mcl of dCTP (2 mM), 3.8 mcl of Cy3-dCTP (2 mM), 4.2 mcl of water, 0.5 mcl of Taq DNA polymerase (5 units/mcl), and 4 mcl of the DNA BAC clone in the volume of the reaction mixture of 30 mcl. The bacteria are cultivated in a Luria Bertani medium with the chloramphenicol antibiotic at a concentration of at least 12 mcg/ml. Plasmid DNA is isolated using columns with a silicate carrier.;EFFECT: invention provides a possibility of obtaining a sufficient concentration of a specific DNA probe for a reaction of fluorescent in situ hybridisation (FISH) with a high brightness of the resulting fluorescent signal, allowing for visual detection of the deletion.;1 cl, 1 tbl, 5 dwg, 1 ex