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

1988/12/01 by Michael A. Innis, K B Myambo, David H. Gelfand +1 · 4 citations
Biochemistry, Genetics and Molecular Biology · #Genomics and Phylogenetic Studies #RNA and protein synthesis mechanisms #Bacterial Genetics and Biotechnology

paper · doi:10.1073/pnas.85.24.9436

openalex publication_date 1988/12/01 · openalex created_date 2025/10/10 · openalex updated_date 2026/07/30

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.

Citations

• Enzymatic Amplification of β-Globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell Anemia
• DNA sequencing with chain-terminating inhibitors

Cited by

• Effective amplification of long targets from cloned inserts and human genomic DNA.
• PCR Bias in Ecological Analysis: a Case Study for Quantitative Taq Nuclease Assays in Analyses of Microbial Communities
• High-level expression, purification, and enzymatic characterization of full-length Thermus aquaticus DNA polymerase and a truncated form deficient in 5' to 3' exonuclease activity.
• Polymerase chain reaction [wikipedia]