Overview
- Template-dependent DNA polymerase
- Catalyzes 5′-3′ synthesis from primed single-stranded DNA
- Possesses 5’→3′ polymerase activity
- Possesses 3′-5′ exonuclease activity
- Lacks 5′-3′ exonuclease activity
Description
- T4 DNA polymerase is a highly processive, DNA-dependent DNA polymerase derived from bacteriophage T4, which infects Escherichia coli. It plays a central role in phage DNA replication and repair. Structurally and functionally distinct from bacterial DNA polymerases, T4 DNA polymerase exhibits robust 5′→3′ DNA polymerase activity along with strong 3′→5′ exonuclease (proofreading) activity, but lacks 5′→3′ exonuclease activity.
- This enzyme is widely used in molecular biology due to its high fidelity and powerful exonuclease function, which allows for precise manipulation of DNA ends. One of its key applications is in the generation of blunt-ended DNA fragments from overhangs by either filling in 5′ overhangs or chewing back 3′ overhangs. This makes it especially useful in preparing DNA fragments for blunt-end ligation in cloning workflows.
- T4 DNA polymerase also plays a role in second-strand cDNA synthesis, site-directed mutagenesis, and other in vitro DNA manipulations where accurate end modification is required. Its high proofreading activity contributes to increased accuracy in applications where base incorporation must be strictly controlled, although this same activity requires careful optimization in reactions where nucleotide analogs or modified bases are present.
- In summary, T4 DNA polymerase is a high-fidelity, exonuclease-rich enzyme that is indispensable for DNA end-processing applications. Its ability to create blunt ends and maintain accuracy during DNA synthesis makes it a powerful tool in recombinant DNA technology and genome engineering protocols.
Applications
- Blunting of DNA ends (fill-in of 5′-overhangs or/and removal of 3′-overhangs)
- DNA labeling (Probe labeling)
