This results in forms of the enzyme being expressed that retain 5' → 3' polymerase activity, but lack any exonuclease activity (5' → 3' or 3' → 5'). This problem can be overcome by introducing mutations in the gene that encodes Klenow. Just as the 5' → 3' exonuclease activity of DNA polymerase I from E.coli can be undesirable, the 3' → 5' exonuclease activity of Klenow fragment can also be undesirable for certain applications. The Klenow fragment was also the original enzyme used for greatly amplifying segments of DNA in the polymerase chain reaction (PCR) process, before being replaced by thermostable enzymes such as Taq polymerase. Filling in receded 3' ends of DNA fragments to make 5' overhang blunt.Synthesis of double-stranded DNA from single-stranded templates.The Klenow fragment is extremely useful for research-based tasks such as: coli makes it unsuitable for many applications, the Klenow fragment, which lacks this activity, can be very useful in research. 5' → 3' polymerase activity, and 3' → 5' exonuclease activity).īecause the 5' → 3' exonuclease activity of DNA polymerase I from E. coli is cleaved by subtilisin retains the 5' → 3' exonuclease activity but does not have the other two activities exhibited by the Klenow fragment (i.e. The other smaller fragment formed when DNA polymerase I from E. First reported in 1970, it retains the 5' → 3' polymerase activity and the 3’ → 5’ exonuclease activity for removal of precoding nucleotides and proofreading, but loses its 5' → 3' exonuclease activity. coli is enzymatically cleaved by the protease subtilisin. The Klenow fragment is a large protein fragment produced when DNA polymerase I from E. Functional domains in the Klenow Fragment (left) and DNA Polymerase I (PDB).
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