Open AccessA systematic method for DNA fragment amplification and sequencing based on DNA indexing technology. Protocol and technical considerations
Author(s) -
Katarzyna A. Gromek,
Tadeusz Kaczorowski
Publication year - 2021
Publication title -
acta biochimica polonica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.452
H-Index - 78
eISSN - 1734-154X
pISSN - 0001-527X
DOI - 10.18388/abp.2020_5737
Subject(s) - subcloning , sequencing by ligation , dna nanoball sequencing , dna sequencing , biology , dna , restriction enzyme , genomic library , computational biology , multiple displacement amplification , plasmid , polymerase chain reaction , genetics , dna extraction , gene , base sequence
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.