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 ). 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. 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). Als Klenow-Fragment, auch Klenow-Enzym, wird das größere der beiden Proteinfragmente der DNA-Polymerase I aus Escherichia coli bezeichnet, die nach enzymatischer Spaltung mit Subtilisin entstehen. 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). Gel elec-trophoretic analyses revealed that the DNA polymerase stalled immediately before the i-motifforming region, indicating that the i-motif is an obstruction to the polymerase. The Klenow fragment and the bacteriophage T4 DNA polymerase are no exceptions as the catalytic efficiency for dATP incorporation is at least 10-fold greater than any other natural nucleotide. The Klenow fragment of DNA polymerase I is a proteolytic fragment obtained by the treatment of DNA polymerase I with subtilisin. cation by Klenow fragment DNA polymerase (KF). This end repair step is followed by 3 A-tailing at 37 C using a mesophilic polymerase such as Klenow Fragment 3-5 exonuclease minus 11, or at elevated temperatures using a thermophilic.