DNA Sequencing by Indexer Walking

Abstract

There is a need for DNA sequencing methods that are faster, more accurate, and less expensive than existing techniques. Here we present a new method for DNA analysis by means of indexer walking. For DNA sequencing by indexer walking, we ligated double-stranded synthetic oligonucleotides (indexers) to DNA fragments that were produced by type IIS restriction endonucleases, which generate nonidentical 4-nucleotide 5' overhangs. The subsequent amplification (30 thermal cycles) of indexed DNA provided a template for automated DNA sequencing with fluorescent dideoxy terminators. The data gathered in the first sequencing reaction permitted further movement into the unknown nucleotide sequence by digestion of analyzed DNA with selected type IIS restriction endonuclease followed by ligation of the next indexer. A library of presynthesized indexers consisting of 256 oligonucleotides was used for bidirectional analysis of DNA molecules and provided universal primers for sequencing. The proposed protocol was successfully applied to sequencing of cryptic plasmids isolated from pathogenic strains of Escherichia coli. The overall error rate for base-calling was 0.5%, with a mean read length of 550 nucleotides. Approximately 1000 nucleotides of high-quality sequence could be obtained per day from a single clone. Indexer walking can be used as a low-cost procedure for nucleotide sequence determination of DNA molecules, such as natural plasmids, cDNA clones, and longer DNA fragments. It can also serve as an alternative method for gap filling at the final stage of genome sequencing projects.