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#3' end

3 public questions tagged with this topic.

Replication Basics

Proofreading occurs at which end of growing strand?

DNA polymerases universally synthesize only 5' to 3' direction adding nucleotides to free 3' hydroxyl of growing primer chain. Proofreading exonuclease activity scans most recently added nucleotide because misincorporation creates mismatch at growing tip distorting duplex geometry and slowing next polymerization step dramatically. Polymerase then reverses one step moving branched 3' terminus from polymerase site to distinct exonuclease site that specifically hydrolyzes phosphodiester bond from 3' end preferentially. Excision cannot occur internally or at 5' terminus restricting surveillance efficiently to active 3' terminus where errors arise during elongation.

Ref: Lodish et al., Molecular Cell Biology, 9th ed., Chapter 11: Proofreading at the 3' terminus during replication

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Restriction Digestion & Mapping

Which end of DNA is typically labeled during restriction mapping?

Restriction mapping tracks fragments originating from a defined terminus to deduce site order, so terminal labeling is essential. Restriction enzymes producing 5' overhangs expose a recessed 3'-OH and a free 5'-phosphate, ideal substrate for T4 polynucleotide kinase to add 32P or for Klenow fragment to fill in with radioactive dNTPs. Internal loci remain phosphodiester-linked without free ends, blunt termini incorporate label poorly, and 3'-only labeling lacks efficiency. Therefore labeling free 5' cohesive ends creates a single radioactive reference point for stepwise fragment analysis.

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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