Nick translation radiolabeling

A sample of total DNA derived from the cells in which the product is produced is then radiolabelled with using the process of nick translation. It is heated to 90°C (promotes... 

The nick-translation method was originally used to incorporate radiolabeled nucleotides into DNA probes. More recent work has shown that nucleotides labeled with biotin can also be incorporated using this method, to yield probes of equivalent sensitivity. Uracil and adenine nucleotides that have been labeled with biotin are shown in Figure 9.18. 

Although [a- 2p]dNTP is generally used as a radiolabel in nick translation, [a- SjdNTP can be used and may have some advantages where detectability is not the primary concern (i) it has a longer half-life (6 X) (ii) shows less radiolysis (iii) tighter bands are obtained (Radford, 1983). The standard nick translation protocol should then be modified since incorporation is much slower... 

Probes radiolabeled by nick translation are usually stable up to 1-2 weeks (depending on specific activity, Section 7.2.2). After nick translation, enzymes are inactivated by adding EDTA to 20 mM DNA can be selectively precipitated with ethanol (Section 3.1.4.1) and, if necessary, using 2.5 pg of carrier tRNA. The precipitate can be dissolved in 1 ml of water. To determine the incorporation of radiolabel, 2 pi of the sample is placed on a glass filter (Whatman... 

Fig. 5. Analysis of RNA complementary to MMTV DNA in 341 cells treated with (ADP-ribose) synthetase inhibitors. Total RNA from cells treated with 10 tM 3-ABm for 0 lanes a, f), 4 (lane b), 16 (lanes c, h), 32 (lane d), and 64 h (lane e), or with 10 mM 3-aminobenzoic acid for 16 h (lane g), was subjected to electrophoresis on 1.2% agarose gels, transferred to a nitrocellulose paper, and hybridized with nick-translated P]-labeled MMTV DNA specific for the env region. The size of the RNA were extrapolated from the migration of radiolabeled Hind III DNA standards (lane i) (4.4, 2.3, and 2.0) kilobase pair (Kbp) and 18S and 28S rRNA from the ethidium bromide staining pattern...

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