The binding sites of KF and T4 DNA polymerase are shown to be quite similar. Ln3+ luminescence spectroscopy is established as a sensitive way to determine the consequences of exonuclease binding-site mutations and to examine binding site similarities and differences among DNA polymerases from different sources. Eu3+ serves as a competitive inhibitor of Mg2+-induced polymerase and exonuclease activity, validating its use as a probe for these active sites. The KF double mutant (D355A, E357A) exhibits a markedly altered and weakened binding site (Kd = 20-26 microM). The single mutants of KF (D424A) and T4 (D219A) also bind a single Eu3+ ion tightly, but the alignment of the coordinating ligands is altered. Both wild-type enzymes tightly bind a single Ln3+ ion but in two isomeric forms. The luminescence spectroscopy observed upon binding of Eu3+ to the exonuclease active site of T4 DNA polymerase was interpreted relative to the binding of Eu3+ or Tb3+ observed with KF. Klenow fragment induced both possible transitions and deletions of 2 and 4 base pairs.The exonuclease active site of the Klenow fragment (KF) of Escherichia coli DNA polymerase I has a double binding site for the two essential divalent metal ions in the presence of the nucleotide monophosphate dTMP. Analyze agarose gels with longwave UV (360 nM) to minimize UV exposure that may cause DNA damage. T4 DNA Polymerase T4 phage replicative polymerase possesses 3 to 5 exo activity. The Quick Blunting Kit ( NEB E1201) is optimized to blunt and phosphorylate DNA ends for cloning in less than 30 minutes. coli DNA Pol I DNA repair polymerase lacks exo. The predominant mutations were sequenced and found to be transitions of G.C to A.T for T4 and modified T7 DNA polymerases, and A.T to G.C for Taq polymerase. T4 DNA Polymerase ( NEB M0203 ) or Klenow ( NEB M0210) will fill in a 5 overhang and chew back a 3 overhang. The error rate for T4 DNA polymerase was not more than 3 x 10(-6) error per base duplication. Therefore, the exonuclease activity for the D219A. ![]() The error rate induced in the 104-base-pair low-temperature melting domain of exon 3 of the human hypoxanthine/guanine phosphoribosyltransferase (HPRT) gene was approximately 3.4 x 10(-5) for modified T7, 1.3 x 10(-4) for Klenow fragment, and 2.1 x 10(-4) for Taq polymerases after a 10(6)-fold amplification. DNA polymerase I (Klenow fragment) (24) have played inte- gral roles in. DNA polymerase I, Klenow fragment, T4 DNA polymerase) have low processivity and dissociate from a primer template after. Removal of a 5' overhang can be accomplished with a nuclease, such as Mung Bean Nuclease. ![]() Incorrectly synthesized sequences were separated from the wild type by DGGE as mutant/wild-type heteroduplexes and the heteroduplex fraction was used to calculate the average error rate (mutations per base duplication). DNA polymerases, such as the Klenow fragment of DNA Polymerase I and T4 DNA Polymerase are often used to fill in (5 3) and chew back (3 5). The strategy permitted direct enumeration and identification of point mutations created by T4, modified T7, Klenow fragment of polymerase I, and Thermus aquaticus (Taq) DNA polymerases. Denaturing gradient gel electrophoresis (DGGE) was used to separate and isolate the products of DNA amplification by polymerase chain reaction (PCR).
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