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

Katarzyna Gromek; Tadeusz Kaczorowski

doi:10.18388/abp.2020_5737

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

Katarzyna Gromek Laboratory of Extremophiles Biology, Department of Microbiology, Faculty of Biology, University of Gdansk, Gdańsk, Poland
Tadeusz Kaczorowski Laboratory of Extremophiles Biology, Department of Microbiology, Faculty of Biology, University of Gdansk, Gdańsk, Poland

DOI: https://doi.org/10.18388/abp.2020_5737

Abstract

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.

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Published

2021-08-26

Issue

Vol. 68 No. 3 (2021): Issue dedicated to Professor Wacław Tadeusz Szybalski on the 100th anniversary of his birth

Section

Articles

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