DNA replication requires DNA Topoisomerase to remove the supercoiling of DNA that accumulates at the end of a growing replication fork. You wish to perform a PCR amplification of a gene that has been provided to you in a 6 kb plasmid vector. Why will you NOT use topoisomerase in your PCR reaction mix?

1
Taq polymerase has innate topoisomerase activity
2
Denaturation step in the PCR protocol precludes formation of supercoils
3
Reaction buffer has a pH that denatures DNA and avoids supercoiling
4
The 5´→ 3´ exonuclease activity of Taq polymerase does not allow supercoiling

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