Klenow fragment reaction specificity

Fig. 28. Synthesis of labeled DNA probes. A Labeled DNA can be generated using different enzymes (Klenow fragment of DNA polymerase or a terminal transferase) to incorporate labeled nucleotides into specific DNA sequences. Probes can be labeled using radioactive nucleotides or nucleotides labeled with an immunogenic molecule such as biotin. B The labeled probe is then hybridized to the target nucleic acid, which is either bound to a membrane or in a tissue section or cell. An antibody is then used to detect the non-radioactively-labeled probe. C The antibody may be conjugated to a fluorescent or chemiluminescent dye, or an enzyme that produces a color reaction. The target nucleic acid is thus visualized.

Biotinylated dUTP can also be used to label DNA probes by a different method, namely random-primed labeling (4). The principle of this method is based on the reannealing of hexadeoxyribonucleotide primers, which have random specificity, to the denatured DNA strands. The DNA to be labeled has to be linearized and denatured before the strands are used as templates in the labeling reaction. The complementary strands are synthesized from the 3 OH termini of the reannealed hexanucleotides by the Klenow fragment of E. coli DNA polymerase I. The primers reanneal at random sites of the template strands, so that the synthesis of the complementary strands is primed at random sites. If one of the deoxyribonucleoside triphosphates present in the reaction mixture is labeled, the newly synthesized strands will become labeled by the incorporation of the labeled nucleotides. The end product of this reaction is a mixture of unlabeled (template) and labeled... 

The 3 -ends are labeled, after specific restriction enzymes have produced a protruding 5 -end, by a fill-in reaction that allows only one label to be introduced (e.g., labeling with dATP with an overhang with 2 Ts should be avoided). Reverse transcriptase or Sequenase are the preferred enzymes for this fill-in although the Klenow fragment has been widely used. The 3 -> 5 exonuclease activity of Klenow, however, may remove the protruding template end. 

Two photoreactive dATP analogues (115, 116) have been incorporated into DNA using Klenow fragment. Subsequent UV irradiation of the primer extension reaction allowed specific cross-linking of the analogues to the polymerase. ... 

The wild-type (25)s insert is purified from the plasmid and end-labeled with Klenow fragment exactly as described for the Southwestern experiment, except that we start out with 50 /xg plasmid to get a final yield of approximately 2 /xg of insert. The 2 jxg of insert is labeled separately in two reactions, as described for Southwestern probe labeling. It is important that the probe has a high specific activity. After purification through Sephadex G50, radioactivity of the probe is measured by scintillation counting. Expect to get at least 2x10 cpm//xl, or 2 x 10 cpm in 100 /xl. Total counts are 4 x 10 in 200 /xl. Add directly to 100 ml hybridization buffer. 

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