Engineered viral DNA polymerase with enhanced DNA amplification capacity: a proof-of-concept of isothermal amplification of damaged DNA

Abstract

AbstractThe development of whole genome amplification (WGA) and related methods, coupled with the dramatic growth of sequencing capacities, has changed the paradigm of genomic and genetic analyses. This has led to a continual requirement of improved DNA amplification protocols and the elaboration of new tailored methods. As key elements in WGA, identification and engineering of novel, faithful and processive DNA polymerases is a driving force in the field. We have engineered the B-family DNA polymerase of virus Bam35 with a C-terminal fusion of DNA-binding motifs. The new protein, named B35-HhH, shows faithful DNA replication in the presence of magnesium or an optimised combination of magnesium and manganese divalent cofactors, which enhances the replication of damaged DNA substrates. Overall, the newly generated variant displays improved amplification performance, sensitivity, translesion synthesis and resistance to salt, which are of great interest for several applications of isothermal DNA amplification. Further, rolling-circle amplification of abasic site-containing minicircles provides a proof-of-concept for using B35-HhH for processive amplification of damaged DNA samples.