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#Klenow fragment

2 public questions tagged with this topic.

Replication in Prokaryotes

Klenow fragment lacks which activity?

Limited proteolysis of Pol I by subtilisin yields large C-terminal Klenow fragment comprising polymerase and 3' to 5' exonuclease domains, removing N-terminal 5' to 3' exonuclease domain. Klenow retains templated synthesis and proofreading, widely used historically for DNA labeling, second strand cDNA synthesis, and Sanger sequencing before recombinant enzymes available. Loss of 5' to 3' activity prevents unwanted degradation of template during synthesis. Modern Klenow exo-minus version also lacks proofreading for higher efficiency incorporation of modified nucleotides and dideoxy terminators.

Ref: Lodish et al., Molecular Cell Biology, 9th ed., Tool Box: Klenow fragment preparation and activities, proteolysis of Pol I

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Restriction Digestion & Mapping

End labeling with Klenow fragment labels which site?

Klenow fragment retains polymerase and 3' to 5' exonuclease but lacks 5' to 3' exonuclease activity, making it ideal for labeling. For end labeling, it fills recessed 3' ends templated by 5' overhangs incorporating alpha-32P labeled nucleotides efficiently. Blunt ends possess recessed 3' terminus after transient breathing and can be labeled by exchange reaction under certain conditions with high deoxynucleotide and enzyme concentrations. While sticky 5' overhangs are preferred substrates for fill-in, blunt-end labeling is classic method to create uniformly labeled probes for restriction mappin

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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