Klenow fragment extension9/28/2023 ![]() ![]() The dNTPs formed from this activity would reduce our concentration of radiolabeled dNTP's, and cause gaps of unlabeled sequences in our new copy. If DNA pol I was used in this example, it could degrade (5'-3' exnuclease) some of these strands (ones that did not have a primer attached). Klenow Fragment (3 5 exo-) is an N-terminal truncation of DNA Polymerase I which retains polymerase activity, but has lost the 5 3 exonuclease activity and has mutations (D355A, E357A) which abolish the 3 5 exonuclease activity (1). The only reasoning I could come up with was, with the use of denaturing dsDNA, there would be free floating ssDNA. However, when it comes to primer extension radio labeling (using a single strand as the template - cDNA, or maybe even denaturing of dsDNA?), the klenow fragment is used.why? It seems when it comes to nick translation radiolabeling, DNA pol I is used understandable since the 5'-3' exonuclease is needed to degrade the non-template strand, allowing for the radiolabeling. My question revolves around radioactive labeling and its applications. Primer extension reactions catalyzed by Klenow fragment (exo+) were. coli is enzymatically cleaved by the protease subtilisin. A single-stranded, 3 extension of three nucleotides bound to the 3 to 5 exonuclease active site. The Klenow fragment is a large protein fragment produced when DNA polymerase I from E. Klenow fragment of Escherichia coli DNA polymerase I. Klenow Fragment is supplied in 50 mM potassium phosphate (pH 6.5), 1 mm DTT, and 50 glycerol (2140A/B/S) or ethylene glycol (2140AK/BK). Degrade 3' over hangs (again it seems DNA pol I could do this, or would the 5'-3' exonuclease begin to degrade the opposite strand?) The Klenow fragment of E.coli DNA polymerase I (exo+), T4 polynucleotide kinase. Klenow fragment Functional domains in the Klenow Fragment (left) and DNA Polymerase I (PDB). Klenow Fragment (DNA Polymerase I Large Fragment) possesses the 5'3' polymerase and 3'5' exonuclease activities of intact DNA Polymerase I, but lacks its 5'3' exonuclease activity. Fill in 5' over hangs (Can't DNA pol I do this?) The extension efficiency was also affected by the up-stream overhanging structure of the primertemplate complex. Many sources say klenow fragments are able to synthesize dsDNA from cDNA (make dsDNA from a single strand) but can't DNA pol I do this? (of course both needing primers).Īdditionally, it is pointed out that Klenow fragments can be used to form blunt ends: Under the condition we conducted, the shortest primers polymerized by Klenow fragment (KF) and Taq DNA polymerase in our experiments were respectively heptamer and octamer. My question though stems from many of the sources I've been scouring. Some more information regarding my question:įrom my understanding, the only difference that a klenow fragment has over DNA polymerase I, is its removal of 5'-3' exonuclease activity (additionally, removal of 3'-5' exonuclease activity if specified). ![]()
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