SNP Discrimination Through "ON/OFF" Switch Of Exo～+ Polymerase

Polymerase with 3' to 5' exonuclease plays an important role in the maintenance of in vivo DNA replication fidelity. The 3' exonuclease activity allows extension of 3' mismatched primers because the 3' mismatched nucleotide is removed. Exo~+ polymerases, together with 3' phosphorothioate-modified mismatched primers, works as an off-switch in DNA polymerization. This suggests that the combination of 3 ' phosphorothioate-modified primers with exo~+ polymerases constituted an "on/off" switch, which allows perfectly matched primers to be extended but not mismatched primers. The "on/off" switch will be developed as a new method for reliable SNP assays.Objective: In this study,we tried to find out the optimization of "on/off" switch in template concentration , the number of the phosphorothioate modification and the polymerases with different fidelities.Method: We used plasmid pDC316 as the sample, and designed a group of 3' phosphorothioate-modified primers which were match or mismatch with the template. The "on/off" switch was operated to explore the optimization in three aspects: template concentration, the number of the phosphorothioate modification and the polymerases with different fidelities.Results: The "on/off" switch mediated by Pfu and single phosphorothioate-modified primers could only stop the mismatch primers mediated extension when the template concentration was 2ng/100μ l or below. And the "on/off" switch mediated by Pfu and double phosphorothioate-modified primers could turn off the mismatch primers...