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The Thermo Scientific DecaLabel DNA Labeling Kit is an advanced system for fast synthesis of radiolabeled DNA probes of high specific activity. It's based on the improved random-primed method developed by Feinberg and Vogelstein (see References 1, 2).
The primary improvement over traditional random-primer kits involves the use of random decamers instead of hexamers to ensure more efficient annealing with DNA at 37°C. In addition, Klenow Fragment, exo- has been genetically engineered to remove exonuclease activity. The improved enzyme does not degrade the labeled probe during reaction and allows high labeling yields even from low template amounts. DNA fragments of any length can be uniformly labeled. Two labeling mixes are provided for flexibility in using either radiolabeled dATP or dCTP.
Random decamers are annealed to a denatured template DNA, and radiolabeled dNTPs are then incorporated into new DNA strands by Klenow Fragment, exo- (see Figure 1 under Supporting Data).
Figure 1. Effect of reaction time and amount of template on the specific radioactivity of a DNA probe.Varying amounts of Lambda DNA/HindIII fragments were labeled with 50 µCi [α-33P]-dCTP (2500 Ci/mmol) according to the protocol supplied with the kit.
A. P. Feinberg, B. Vogelstein, A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Biochem. 132, 6-13 (1983).
A. P. Feinberg, B. Vogelstein, A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity, Addendum. Biochem. 137, 266-267 (1984).