Rapid cycle sequencing in an air thermal cycler.
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.
Swerdlow
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H
University of Utah, Salt Lake City 84112.
Dew-Jager
K
K
Gesteland
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RF
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
D017931:DNA Primers; D005456:Fluorescent Dyes; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase