Reduced amplification by phi29 DNA polymerase in the presence of unbound oligos during reaction in RCA

Synthetic single-stranded oligonucleotides play crucial roles in DNA amplification reactions for various applications, such as serving as primers, enabling magnetic separation, and generating dsDNA for subsequent digestion. Typically, these oligos are added in excess to ensure rapid binding to their intended targets. However, while performing rolling circle amplification (RCA) using phi29 DNA polymerase, we observed a decrease in amplification efficiency when oligos were present in the reaction. This phenomenon was consistently observed in two separate laboratories, prompting this study to delve into the root causes responsible for the decline in RCA efficiency. The lowered efficiency was consistent regardless of the manufacturer or any mutations in the phi29 polymerase. We identified several variables that influenced RCA efficiency, mainly the length of the oligos used and the presence of modifications, particularly those obstructing 3’ end digestion. This strongly suggests that the exonuclease domain of phi29 DNA polymerase is responsible for the competition-based inhibition. Our investigation shows that even picomole quantities of oligos can significantly reduce total DNA production during the phi29 DNA polymerase-mediated amplification process. Conversely, the addition of oligos to the reaction did not impede the efficiency of Bst 3.0 polymerase, likely due to the lack of an exonuclease domain of said polymerase. While increasing the quantity of phi29 DNA polymerase in the reaction partially alleviated the adverse effects of excess oligos, we believe it is crucial to carefully optimize the oligo quantities to achieve maximum amplification of the desired targets. © 2024 The Author(s)