Rapid cycle sequencing in an air thermal cycler

Biotechniques. 1993 Sep;15(3):512-9.

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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Base Sequence
  • Capillary Action
  • DNA Primers
  • DNA-Directed DNA Polymerase
  • Fluorescent Dyes
  • Hot Temperature
  • Molecular Sequence Data
  • Polymerase Chain Reaction
  • Sequence Analysis, DNA / methods*
  • Taq Polymerase
  • Zebrafish / genetics

Substances

  • DNA Primers
  • Fluorescent Dyes
  • Taq Polymerase
  • DNA-Directed DNA Polymerase