![]() As in the case with the kinase labeling of DNA ( Chapter 39), the DNA fragment is cleaved with a second restriction enzyme, to generate fragments of unequal size that are labeled only in one end.The fragments are subsequently separated on the basis of their different molecular weights and isolated ( chapter 7 and chapter 10). coli polymerase I and the Taq DNA polymerase of Thermus aquaticus, are known to add bases to the 3 9 hydroxyls of blunt-ended DNA duplexes in vitro (29, 30). The DNA fragment to be labeled is incubated in the presence of one or more deoxyribonucleotide triphosphates (one of which is labeled with 32P in the a-phosphate group) and the Klenow fragment of DNA polymerase. In addition, fluorescence restoration was increased through a polymerization reaction by the Klenow fragment in the presence of a fluorescein-attached. The principle of the method is shown in Fig. We propose a model for template recognition with DNA polymerase based on the electrostatic contact and hydrogen bond only of the inter- nucleotide phosphates. The method described here is for generating 3′ end-labeled DNA fragments that are suited for sequencing by the method of Maxam and Gilbert ( Chapter 51), but can in principle also be used when radioactively labeled DNA is required for other purposes (e.g., Southern hybridization). Radiolabeling of DNA fragments is most efficient with DNA fragments that contain recessed 3′ ends ( see Fig. ![]() The fragment still has the polymerase and 3′–5′ exonuclease activity, but lacks the 5′–3′ exonuclease activity of the holoenzyme ( 1). The Klenow fragment of DNA polymerase I is a proteolytic fragment obtained by the treatment of DNA polymerase I with subtilisin. ![]()
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