Efficient PCR Production of Single-Stranded DNA Sequencing Templates

  • Bernhard Kaltenboeck
  • Konstantin G. Kousoulas
Part of the Methods in Molecular Biology™ book series (MIMB, volume 65)


The acceptance of polymerase chain reaction (PCR) as a routine method in molecular biology has created a need for simple and robust approaches to DNA sequencing of the amplification products. In addition to its ease, direct sequencing of PCR products has the advantage that nucleotide misincorporation during amplification does not pose a problem, because only a small percentage of the DNA molecules may contain such random mutations. However, a large body of published protocols attests to the difficulties encountered in this endeavor. Double-stranded sequencing of the linear PCR products is notoriously unreliable because of rapid reannealing of the denatured DNA.


Polymerase Chain Reaction dNTP Concentration dsDNA Fragment Asymmetric Polymerase Chain Reaction ssDNA Sequencing 
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Copyright information

© Humana Press Inc., Totowa, NJ 1996

Authors and Affiliations

  • Bernhard Kaltenboeck
    • 1
  • Konstantin G. Kousoulas
    • 2
  1. 1.Department of PathobiologyCollege of Veterinary Medicine, Auburn University
  2. 2.School of Veterinary MedicineLouisiana State UniversityBaton Rouge

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