DNA sequence analysis with a modified bacteriophage T7 DNA polymerase.

TLDR

A chemically modified phage T7 DNA polymerase is ideal for chain‑termination sequencing due to three key properties. The modified enzyme is highly processive, lacks 3′→5′ exonuclease activity, and efficiently incorporates nucleotide analogs to improve electrophoretic resolution. It produces uniformly intense, background‑free dideoxynucleotide fragments and, with dITP substitution, eliminates band compression for clear single‑orientation sequence determination.

Abstract

A chemically modified phage T7 DNA polymerase has three properties that make it ideal for DNA sequencing by the chain-termination method. The enzyme is highly processive, catalyzing the polymerization of thousands of nucleotides without dissociating. By virtue of the modification the 3' to 5' exonuclease activity is eliminated. The modified polymerase efficiently uses nucleotide analogs that increase the electrophoretic resolution of bands in gels. Consequently, dideoxynucleotide-terminated fragments have highly uniform radioactive intensity throughout the range of a few to thousands of nucleotides in length. There is virtually no background due to terminations at pause sites or secondary-structure impediments. Processive synthesis with dITP in place of dGTP eliminates band compressions, making possible the unambiguous determination of sequences from a single orientation.