Thermostable enzymes represent a promising approach to decreasing the cost of sequencing genomes. Shotgun sequencing strategies, and greatly improved methods of sequencing based on the automated fluorescent DNA sequencers, all have helped increase throughput for whole genome sequencing. For automated fluorescent DNA sequencing, the ability to sequence double stranded DNA reliably would make the process of DNA sequencing more efficient by eliminating the steps of subcloning into M13 vectors. A major problem associated with double-stranded DNA sequencing appears to be related to maintaining the dissociation of the two rapidly reannealing complementary strands while allowing annealing and extension of the sequencing primer. The reassociation of denatured DNA strands results in fewer readable bases. The preliminary experiments indicate sequencing of covalently closed circular DNA is more robust and accurate when sequenced in the presence of thermostable DNA-relaxing topoisomerases. The effect is based on the topological destabilization (unwinding) of the double helix by these enzymes in a wide range of ionic conditions (Slesarev et al. Nature 364:735-7, 1993). Unwinding supercoiled DNA by thermostable topoisomerases appears to improve DNA as a sequencing template by decreasing the number of prematurely terminated molecules which compete with true dideoxy terminations. This reduces background bands and increases the accuracy of base calling. In addition, it is thought that hairpin structures in the template are relieved, eliminating the """"""""strong stop"""""""" artifact. Further, improved denaturation seems to increase the efficiency of primer annealing, increasing the sequencing signal. The conclusions will be presented in the form of comparison of traditional and new methods of DNA sequencing with respect to the fidelity of sequencing, signal intensity and DNA consumption.
| Slesarev, A I; Belova, G I; Lake, J A et al. (2001) Topoisomerase V from Methanopyrus kandleri. Methods Enzymol 334:179-92 |
