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. Ī detailed analysis of solid-state structures of several unrelated systems that catalyze the hydrolysis of diphosphate esters ( alkaline phosphatase and the Klenow fragment, among others) revealed that their active sites invariably contain conserved carboxylate. Įxtensive kinetic experiments on the Klenow fragment (and other replicases) have resulted in the general mechanism illustrated in Figure Except for variations. Cytotoxicity studies in their primary cells and in a T- cell line reveal that efavirenz has a selectivity index of 80,000. The polymerase enzymes studied were Moloney murine leukemia virus RT, human DNA polymerases a, 3, and 7, Escherichia coli RNA polymerase, and the Klenow fragment. Results from testing against a variety of polymerase enzymes show that efavirenz is inactive up to 300 p,M for a 50% inhibition (Young et al., 1995). If these enzymes must be used, an exonuclease such as DNA Polymerase 1 Large (Klenow) Fragment can be utilized to convert the overhang to a blunt end before the template is transcribed. The use of such enzymes has been reported to result in the production of additional, nonspecific transcripts (Schenborn and Mierendorf, 1985). It is important to note that restriction enzymes producing 3 -overhangs should be avoided if possible (e.g., Psfl, Sfil, Kpnl). īiolabs (Pickering, ON), and Promega (Madison, WI). Again if the reaction is carried out in the presence of labelled deoxynucleotides the result is a strand of DNA which is radioactively labelled. It uses the Klenow fragment, which fills in the sticky ends by synthesizing the complementary strand. Įnd filling is a more gentle method but can only be used on DNA molecules that have sticky ends. It is used to create blunt ends in dsDNA and in the dideoxy method of DNA sequencing. This enzyme sythesizes a new DNA strand complementary to the single strand of DNA (the template) only. The part which retains the polymerase function is known as the Klenow fragment. These two parts can be separated by treatment with the enzyme sub-tilisin. The DNA polymerase 1 molecule contains its polymerase and nuclease activities on different parts of the enzyme molecule. The Klenow fragment is sold commercially for use in labeling DNA for use in detecting recombinant DNA. The small fragment retains the 5 to 3 nuclease activity, whereas the larger piece, called a Klenow fragment, has both polymerase activity and the 3 to 5 exonuclease activity. When the pure enzyme is treated with subtilisin, a proteolytic enzyme from Bacillus subtilis, the polymerase is cleaved into two pieces. DNA polymerase I has been purified to homogeneity.
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