Single Stranded Transposon Insertion

Transposase is an enzyme that catalyzes the cleavage and insertion of segments of DNA from one location on the genome to another. Transposase has an application in next-generation sequencing (NGS) library preparation in a... [ view full abstract ]

Transposase is an enzyme that catalyzes the cleavage and insertion of segments of DNA from one location on the genome to another. Transposase has an application in next-generation sequencing (NGS) library preparation in a process called tagmentation, where two transposases are assembled with end sequences (ES) only but no linking donor DNA. This creates a double-stranded DNA break with the ES at the ends of the resulting DNA fragments. Tagmentation activity can be increased by replacing the wild type ES with a synthetic mosaic end sequence (MES). This hyperactive variant of the Tn5 transposase is commonly used in NGS library preparation to both tag and fragment (tagment) double-stranded DNA. In an effort to better understand the mechanisms of Tn5 insertion, we studied the ability of transposase to tagment only a single DNA strand when chemical modifications were added to the 3’ end of one of the complex's MES. We found that the addition of either a phosphate or dideoxy nucleotide can prevent complete tagmentation and results in one MES tagged DNA strand and one intact, unmodified DNA strand. This approach may allow us to block specific steps of transposase enzymatic activity, which can be used to improve our understanding of the exact mechanisms of Tn5 transposase insertion events. Furthermore, this "asymmetrical" tagmentation could be useful in the development of new library preparation methods.