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 DNA polymerase I (KF) including binding, moving, and dissociation from the. The DNA fragment to be labeled is incubated in the presence of one or more deoxyribonucleotide triphosphates (one of which is labeled with (32)P in the a-phosphate group) and the Klenow fragment of DNA polymerase. We imaged several functional activities of the Klenow fragment of E. Elucidation of the metal-binding properties of the Klenow fragment of Escherichia coli polymerase I and bacteriophage T4 DNA polymerase by lanthanide(III) luminescence spectroscopy. The principle of the method is shown in Fig. 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. co- li DNA polymerase I Klenow fragment 25. The fragment still has the polymerase and 3'-5' exonuclease activity, but lacks the 5'-3' exonuclease activity of the holoenzyme (1). been subsequently reported to in- hibit the progression of both T7 bacteriophage DNA polymerase and E. The Klenow fragment of DNA polymerase I is a proteolytic fragment obtained by the treatment of DNA polymerase I with subtilisin. A mechanism by which the Klenow fragment of DNA polymerase I monitors the geometry of the base pairs may involve hydrogen bonds between the polymerase and the minor groove of the nascent base pair.
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