CircumVent thermal cycle sequencing and alternative manual and automated DNA sequencing protocols using the highly thermostable VentR (exo-) DNA polymerase

• Published in: BioTechniques Vol. 13; no. 4; p. 626
• Main Authors: Sears, L E, Moran, L S, Kissinger, C, Creasey, T, Perry-O'Keefe, H, Roskey, M, Sutherland, E, Slatko, B E
• Format: Journal Article
• Language: English
• Published: England 01.10.1992
• Subjects: Autoradiography; DNA - analysis; DNA-Directed DNA Polymerase; Electrophoresis, Polyacrylamide Gel; Sequence Analysis, DNA - methods
• ISSN: 0736-6205

CircumVent thermal cycle and standard DNA sequencing protocols utilizing the cloned and highly thermostable VentR (exo-) DNA polymerase are described. The thermal cycle sequencing procedures are advantageous because they allow fast and simple semiautomation of the sequencing reaction; make possible the direct DNA sequencing of PCR products, bacterial colonies and phage plaques; require only femtomoles of template DNA; eliminate the requirement of an independent primer annealing step; remove the requirement of denatured plasmids for sequencing double-stranded templates; and use a highly thermostable DNA polymerase for sequencing through potential recalcitrant secondary structure domains and large linear double-stranded DNA templates such as lambda derivatives. More standard methods of DNA sequencing (i.e., a one-step protocol and a labeling-termination protocol) are also presented. For each protocol, alternatives for choice of label and method of labeling are presented, including the use of 5' biotinylated primers for chemiluminescent DNA sequencing and fluorinated primers for automated sequencing using the BaseStation Automated DNA Sequencer.