DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA.

TLDR

The Thermus aquaticus DNA polymerase (Taq) is highly thermostable, fast, processive, and lacks 3′‑exonuclease activity, making it ideal for DNA sequencing across a wide temperature range. The authors present protocols that generate >1,000‑base readable extension products with uniform band intensities, using high temperatures and 7‑deaza‑2′‑deoxyguanosine to sequence GC‑rich DNA and resolve gel compressions, and they modify PCR conditions to allow direct sequencing of asymmetric PCR products without purification. Combining asymmetric PCR template preparation with direct sequencing should enable automation for large‑scale sequencing projects.

Abstract

The highly thermostable DNA polymerase from Thermus aquaticus (Taq) is ideal for both manual and automated DNA sequencing because it is fast, highly processive, has little or no 3'-exonuclease activity, and is active over a broad range of temperatures. Sequencing protocols are presented that produce readable extension products greater than 1000 bases having uniform band intensities. A combination of high reaction temperatures and the base analog 7-deaza-2'-deoxyguanosine was used to sequence through G + C-rich DNA and to resolve gel compressions. We modified the polymerase chain reaction (PCR) conditions for direct DNA sequencing of asymmetric PCR products without intermediate purification by using Taq DNA polymerase. The coupling of template preparation by asymmetric PCR and direct sequencing should facilitate automation for large-scale sequencing projects.