Rapid one-step automated sequencing reactions for 16 DNA samples using taq polymerase and fluorescent primers

• Published in: Nucleic acids research Vol. 19; no. 1; p. 188
• Main Authors: CIORA, T, DENEFLE, P, MAYAUX, J.-F
• Format: Journal Article
• Language: English
• Published: Oxford Oxford University Press 11.01.1991
• Subjects: Base Sequence; Biological and medical sciences; Biotechnology; DNA - genetics; DNA - metabolism; DNA-Directed DNA Polymerase - metabolism; Fluorescence; Fundamental and applied biological sciences. Psychology; Genetic engineering; Genetic technics; Genetic Techniques; Methods. Procedures. Technologies; Synthetic digonucleotides and genes. Sequencing; Taq Polymerase; Templates, Genetic; Automation; DNA polymerase; Nucleotide sequence; Enzyme; Process improvement; Primer; Fluorescence; Method
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/19.1.188

We describe a very simple and reliable protocol which also solves all the evaporation problems encountered for sequencing reactions in microtiter plate. We used the property of the thermostable polymerase from Thermophillus aquaticus , Taq, which is optimally active for DNA sequencing at high temperatures (60 to 80 degree c) in order to perform in a single heating step both primer-annealing and polymerization reactions. Hereafter a protocol using the Biomek super(TM) 1000 workstation equipped with the HCB-1000 thermocontroller (Beckman), which allows to perform simultaneous sequencing reactions with fluorescent primers on 16 different single-stranded DNAs in a microtiter plate, is described.