A systematic method for DNA fragment amplification and sequencing based on DNA indexing technology. Protocol and technical considerations

DNA indexing is based on a presynthesized library of oligonucleotide adaptors (256 in total), named indexers, and type-IIS restriction endonucleases. It enables amplification and direct analysis of large DNA fragments with low overall redundancy and without subcloning. Here, we describe a detailed protocol for PCR-based amplification of DNA fragments followed by DNA sequencing by indexer walking and provide helpful hints on its practical use. The proposed protocol can be applied to the sequencing of plasmids, cDNA clones, and longer DNA fragments. It can also be used for gap filling at the final stage of genome sequencing projects.

Automated summary

DNA indexing is a method for amplification and direct analysis of large DNA fragments using a library of oligonucleotide adaptors and type-IIS restriction endonucleases. It can be applied to sequence plasmids, cDNA clones, and longer DNA fragments, as well as fill gaps in genome sequencing projects at the final stage.