title: Rapid cycle sequencing in an air thermal cycler.
abstract: Cycle sequencing using Taq DNA polymerase has gained popularity recently due to reduced template requirements, improved signal and its ability to directly sequence PCR fragments. A major drawback to the technique is the time required for performing reactions in a block-based thermal cycler. To help cycle sequencing compete with other methods, we have modified the protocol to be performed in capillaries using an air-based thermal cycling instrument. This instrument has been developed and optimized for rapid, specific amplification of DNA by PCR. The resulting cycle sequencing methodology is faster than block-based approaches; a reaction can be completed in 25 min, compared with about 2 h in a conventional instrument. Thus, the speed of the technique is competitive with standard uncycled T7 or Taq reactions. Accuracy of the sequencing data is improved; two problem areas in the sequence obtained with a block cycler are ameliorated by the capillary methodology. This technique represents a novel approach to cycle sequencing that will further the development of capillary-based analytical methods.
authors.affiliation: University of Utah, Salt Lake City 84112.
authors.forename: H
authors.initials: H
authors.lastname: Swerdlow
authors.forename: K
authors.initials: K
authors.lastname: Dew-Jager
authors.forename: R F
authors.initials: RF
authors.lastname: Gesteland
mesh_terms: D000818:Animals; D001483:Base Sequence; D002197:Capillary Action; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase; D005456:Fluorescent Dyes; D006358:Hot Temperature; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*; D019914:Taq Polymerase; D015027:Zebrafish / Q000235:genetics
chemical_list: D017931:DNA Primers; D005456:Fluorescent Dyes; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase