"Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase". "Enzymatic Amplification of β-globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell Anemia". "Selective Elimination of the Exonuclease Activity of the Deoxyribonucleic Acid Polymerase from Escherichia coli B by Limited Proteolysis". The exo-Klenow fragment is used in some fluorescent labeling reactions for microarray.ġ. This form of the enzyme is called the exo- Klenow fragment. DNA Polymerase I Large (Klenow) Fragment is a 68kDa C-terminal fragment of E. 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 (meaning removal of overhangs to create blunt ends) recessed 3' ends of DNA fragments * Synthesis of double-stranded DNA from single-stranded templates Here, let’s look at some more differences between the Klenow Fragment and DNA Polymerase. It lacks the 5’ to 3’ exonuclease domain of the DNA polymerase. The Klenow fragment is extremely useful for research-based tasks such as: NEET 2023 Answer Key Difference between Klenow Fragment and DNA Polymerase Klenow fragment is the largest segment of DNA polymerase. 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).įunctional domains in the Klenow Fragment (left) and DNA Polymerase I (PDB).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. The polymerase domain consists of three subdomains: the fingers, the palm, and the thumb. coli is enzymatically cleaved by the protease subtilisin. coli DNA polymerase I (Klenow fragment KF), an active truncated form of polymerase I, is composed of two domains: a polymerase domain that incorporates nucleotides, and a 3-5 exonuclease domain that excises misincorporated nucleotides. The Klenow fragment is a large protein fragment produced when DNA polymerase I from E.
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