Chimeric DNA byproducts in strand displacement amplification using the T7 replisome

Recent advances in next generation sequencing technologies enable reading DNA molecules hundreds of kilobases in length and motivate development of DNA amplification methods capable of producing long amplicons. In vivo, DNA replication is performed not by a single polymerase enzyme, but multiprotein complexes called replisomes. Here, we investigate strand-displacement amplification reactions using the T7 replisome, a macromolecular complex of a helicase, a single-stranded DNA binding protein, and a DNA polymerase. The T7 replisome may initiate processive DNA synthesis from DNA nicks, and the reaction of a 48 kilobase linear double stranded DNA substrate with the T7 replisome and nicking endonucleases is shown to produce discrete DNA amplicons. To gain a mechanistic understanding of this reaction, we utilized Oxford Nanopore long-read sequencing technology. Sequence analysis of the amplicons revealed chimeric DNA reads and uncovered a connection between template switching and polymerase exonuclease activity. Nanopore sequencing provides insight to guide the further development of isothermal amplification methods for long DNA, and our results highlight the need for high-specificity, high-turnover nicking endonucleases to initiate DNA amplification without thermal denaturation.

Automated summary

Recent advances in next generation sequencing technologies have made it possible to read DNA molecules hundreds of kilobases in length. This has motivated the development of DNA amplification methods capable of producing long amplicons. In this study, the researchers investigated strand-displacement amplification reactions using the T7 replisome, a multiprotein complex involved in DNA replication. They utilized Oxford Nanopore long-read sequencing technology to gain insight into the reaction mechanism. The results revealed chimeric DNA reads and a connection between template switching and polymerase exonuclease activity.