Strategies for rapid and accurate DNA sequencing

In an effort to increase the amount of accurate final sequence data, we now report our latest template DNA isolation procedure, fluorescent and radiolabeled DNA sequencing reactions, physical modifications to the ABI 373A fluorescent-based DNA sequencer, and the development of a new fluorescent data analysis program that results in manually interpretable sequence data in excess of 750 nucleotides from the priming site in a single reaction. In addition, a sequencing strategy that combines these isolation and sequencing methods with initial shotgun cloning followed by a short oligonucleotide primer walking approach for both contig joining and proofreading is described. When combined, these methods and strategies significantly increase the rate and ease of DNA sequence data acquisition, as well as improve the extent and accuracy of the sequence data.