We examined complexes of Klenow fragment with DNAs containing various base mismatches. Each batch of random oligonucleotides contains all possible sequences (for hexamers, which are most commonly employed, this would be 4096 different oligonucleotides) and so any DNA template can be used with this method. The time-resolved motions of the dansyl probes were sensitive indicators of DNA-protein contacts, showing that the protein binds to DNA with two footprints, corresponding to primer termini at either the polymerase or 3'-5' exonuclease sites. Klenow fragment polymerase is then used to extend the oligonucleotides, using three cold nucleotides and one radioactively labeled nucleotide provided in the reaction mixture, to produce a uniformly labeled double-stranded probe. coli DNA Polymerase I which retains polymerization and 3 5 exonuclease activity, but has lost 5 3 exonuclease activity (1). Then, random sequence oligonucleotides are annealed to both strands. coli DNA polymerase I, large fragment) produced the same amplification patterns as Bsu and the maximum activity was observed at approximately 38 C (Supplemental Fig. DNA Polymerase I, Large (Klenow) Fragment is a proteolytic product of E. The following method is essentially that described by Feinberg and Vogelstein (2) in which a DNA fragment is denatured by heating in a boiling water bath. The crystal structure showed that the fragment is folded into two distinct domains. ![]() Random primed labeling can give specific activities of between 2 × 10(9) and 5 × 10(9) dpm/μg (see Note 1). The Klenow fragment of DNA polymerase I from Escherichia coli has two enzymatic activities: DNA polymerase and 3-5 exonuclease. ![]() Random primed labeling, based on the method of Feinberg and Vogelstein (1) is a method of incorporating radioactive nucleotides along the length of a fragment of DNA. Random primed labeling of DNA has now almost superseded the method of nick translation of DNA.
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