Genomic Sequencing with Taq Polymerase (Linear Amplification)

Abstract

The use of Taq polymerase for the linear amplification of genomic DNA fragments by chemical sequencing reactions (Saluz and Jost, 1989) retains all the benefits of the classical procedure (Church and Gilbert, 1984), while being radically simplified. The scheme of this procedure is shown in the flow diagram (Fig. IV. 1). Total genomic DNA is digested with a suitable restriction enzyme to reduce its viscosity. The resulting DNA fragments are chemically sequenced as described by Maxam and Gilbert (1980), Fritzsche et al. (1987), or Rubin and Schmidt (1980). Sequencing is followed by selective linear amplification with a primer labeled to a very high specific radioactivity by the filling-in procedure using the T7 DNA polymerase (Sequenase version 2; produced from recombinant DNA; United States Biochemical, USB). The linear amplification of the genomic DNA fragments is performed with the thermostable Taq polymerase. The amplification products are then purified and separated on a sequencing gel. The sequence information is obtained by exposing the dried gel to an X-ray film.