The Klenow fragment is a large protein fragment produced when DNA polymerase I from E. coli is enzymatically cleaved by the proteasesubtilisin. 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.
Functional domains in the Klenow Fragment (left) and DNA Polymerase I (PDB).
The other smaller fragment formed when 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. 5' → 3' polymerase activity, and 3' → 5' exonuclease activity).
Because the 5' → 3' exonuclease activity of DNA polymerase I from E. 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:
Synthesis of double-stranded DNA from single-stranded templates
Filling in receded 3' ends of DNA fragments to make 5' overhang blunt
The Klenow fragment was also the original enzyme used for greatly amplifying segments of DNA in the polymerase chain reaction (PCR) process,[2] before being replaced by thermostable enzymes such as Taq polymerase.[3]
The exo- Klenow fragmentedit
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. This problem can be overcome by introducing mutations in the gene that encodes Klenow. 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 form of the enzyme is called the exo- 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])
Referencesedit
^Klenow H and Henningsen I (1970). "Selective Elimination of the Exonuclease Activity of the Deoxyribonucleic Acid Polymerase from Escherichia coli B by Limited Proteolysis". Proc. Natl. Acad. Sci. USA. 65 (1): 168–175. Bibcode:1970PNAS...65..168K. doi:10.1073/pnas.65.1.168. PMC286206. PMID 4905667.
^Saiki RK, et al. (1985). "Enzymatic Amplification of β-globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell Anemia". Science. 230 (4732): 1350–54. Bibcode:1985Sci...230.1350S. doi:10.1126/science.2999980. PMID 2999980.
^Saiki; et al. (1988). "Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase". Science. 239 (4839): 487–91. doi:10.1126/science.239.4839.487. PMID 2448875.
klenow, fragment, large, protein, fragment, produced, when, polymerase, from, coli, enzymatically, cleaved, protease, subtilisin, first, reported, 1970, retains, polymerase, activity, exonuclease, activity, removal, precoding, nucleotides, proofreading, loses,. The Klenow fragment is a large protein fragment produced when DNA polymerase I from E coli is enzymatically cleaved by the protease subtilisin 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 Functional domains in the Klenow Fragment left and DNA Polymerase I PDB The other smaller fragment formed when 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 5 3 polymerase activity and 3 5 exonuclease activity Contents 1 Research 2 The exo Klenow fragment 3 References 4 External linksResearch editBecause the 5 3 exonuclease activity of DNA polymerase I from E 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 Synthesis of double stranded DNA from single stranded templates Filling in receded 3 ends of DNA fragments to make 5 overhang blunt Digesting away protruding 3 overhangs Preparation of radioactive DNA probesThe Klenow fragment was also the original enzyme used for greatly amplifying segments of DNA in the polymerase chain reaction PCR process 2 before being replaced by thermostable enzymes such as Taq polymerase 3 The exo Klenow fragment editJust 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 This problem can be overcome by introducing mutations in the gene that encodes Klenow 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 form of the enzyme is called the exo 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 References edit Klenow H and Henningsen I 1970 Selective Elimination of the Exonuclease Activity of the Deoxyribonucleic Acid Polymerase from Escherichia coli B by Limited Proteolysis Proc Natl Acad Sci USA 65 1 168 175 Bibcode 1970PNAS 65 168K doi 10 1073 pnas 65 1 168 PMC 286206 PMID 4905667 Saiki RK et al 1985 Enzymatic Amplification of b globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell Anemia Science 230 4732 1350 54 Bibcode 1985Sci 230 1350S doi 10 1126 science 2999980 PMID 2999980 Saiki et al 1988 Primer directed enzymatic amplification of DNA with a thermostable DNA polymerase Science 239 4839 487 91 doi 10 1126 science 239 4839 487 PMID 2448875 External links editKlenow Fragment at the U S National Library of Medicine Medical Subject Headings MeSH Diagram at vivo colostate edu Retrieved from https en wikipedia org w index php title Klenow fragment amp oldid 1052913469, wikipedia, wiki, book, books, library,
