DNA sequencing using Taq polymerase.

Three DNA polymerases, namely E.coli DNA polymerase 1 (Klenow), reverse transcriptase and T7 DNA polymerase (sequenase), are commonly used for DNA sequencing by the chain termination method of Sanger and colleagues [1). However, the secondary structure of the DNA template can impede the progress of all three polymerases. I have developed a novel procedure in which the thermostable polymerase of Thermus aquaticus [2] (Taq polymerase) is utilized in a reaction incorporating 7-deaza dGTP and performed at 70°C, at which the effects of secondary structure are eliminated. The procedure involved a 2 step reaction: 1) Labeling reaction. In a single tube the universal M13 primer was annealed to the M13 template and extended in the presence of a limiting amount of all 4 deoxynucleotides, the dATP being radiolabelled with either 32p or 35S; 2) Termination reaction. The product was divided into 4 tubes, each containing the appropriate deoxy/dideoxynucleotide mixes. Fig.1 shows a comparison of this sequencing procedure with that obtained using the sequenase kit supplied by United States Biochemicals. The single-stranded M13 template used to test this procedure was a fragment of a cDNA encoding 1,3,1,4, B-gluconase from wheat, which contains a region with strong secondary structure that cannot be unambiguously sequenced on either strand using sequenase. With sequenase at 37°C, strong stops were seen in all 4 tracks at the position marked by the arrow. For a few bases above and below this position weaker bands were seen in all 4 tracks. Sequencing this template with Taq polymerase at 70°C resulted in an unambiguous reading through this region. The strong stop observed with sequenase occured at a G residue in the middle of an extremely G/C rich area which has the potential to form a hairpin structure. As well as being able to reveal the sequence of DNA templates with strong secondary structure, Taq polymerase has further advantages over sequenase. Firstly, a separate annealing reaction is not necessary, (which simplifies the protocol), secondly, Taq polymerase is more stable than sequenase which allows all the constituents of the labeling reaction (except the template) to be mixed before aliquoting, and thirdly Taq polymerase is cheaper by a factor of 4:1.