[Development of the method of RNA labeling in vitro using the terminal transferase with the purpose to obtain hybridization probes].
A possibility to use terminal transferase to obtain hybridized RNA-probes has been investigated. Optimal conditions of the reaction proceeding were elaborated on the model objects--RNA of the phage MS2: 200 mM of potassium cacodylate, pH 7.2, 1 mM CoCl2, 1 mM DTT, under the considerable surplus of the enzyme. The elaborated method was used to determine the area of early genes of cyanophage LPP-3. With this purpose total RNA preparations were isolated from cyanobacterium Plectonema boryanum CALU 465. The above preparations were labeled by means of terminal transferase and [alpha-32P] ATP and used as the probes in hybridization with the DNA restriction fragments of LPP-3. Proceeding from the results of Southern-blots, a conclusion was made, that the KpnI-fragment of genome of cyanophage LPP3 10 kb in size includes the early genes. A possibility of practical use of the developed method is discussed.
Syrchin
S A
SA
Institute of Microbiology and Virology, National Academy of Sciences of Ukraine, 154, Zabolotny St., Kyiv, 03143, Ukraine.
Mendzhul
M I
MI
D007553:Isotope Labeling; D009693:Nucleic Acid Hybridization / Q000379:methods*; D010761:Phosphorus Radioisotopes; D015347:RNA Probes; D014166:Transferases
D010761:Phosphorus Radioisotopes; D015347:RNA Probes; D014166:Transferases
Razrabotka metoda mecheniia RNK in vitro s pomoshch'iu terminal'noĭ transferazy s tsel'iu polucheniia gibridizatsionnykh zondov.