TheKlenow fragment is a largeprotein fragment produced whenDNA polymerase I fromE. coli isenzymatically cleaved by theproteasesubtilisin. First reported in 1970,[1] 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 other smaller fragment formed when DNA polymerase I fromE. 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. 5' → 3' polymerase activity, and 3' → 5' exonuclease activity).
Because the 5' → 3' exonuclease activity of DNA polymerase I fromE. coli makes it unsuitable for many applications, the Klenow fragment, which lacks this activity, can be very useful in research. The Klenow fragment is extremely useful for research-based tasks such as:
The Klenow fragment was also the original enzyme used for greatly amplifying segments of DNA in thepolymerase chain reaction (PCR) process,[2] before being replaced bythermostable DNA polymerases such asTaq polymerase.[3]
Just as the 5' → 3'exonuclease activity ofDNA polymerase I fromE.coli can be undesirable, the 3' → 5'exonuclease activity of Klenow fragment can also be undesirable for certain applications. This problem can be overcome by introducingmutations in the gene that encodes Klenow. This results in forms of the enzyme being expressed that retain 5' → 3' polymerase activity, but lack anyexonuclease activity (5' → 3' or 3' → 5'). This form of the enzyme is called theexo-Klenow fragment.
The exo-Klenow fragment is used in some fluorescent labeling reactions for microarray, and also in dA and dT tailing, an important step in the process of ligating DNA adapters to DNA fragments, frequently used in preparing DNA libraries for Next-Gen sequencing. (for instance see[1])