Primer Extension Reaction Assays for Incorporation of Deoxynucleotide Analogue into DNA

Abstract

Incorporation of deoxynucleotide analogues into DNA is important for the expansion of DNA functions. Primer extension reactions are commonly used for the assay of such reaction events. However, current assay protocols generally rely on radiolabeling, fluorescence reporter labeling, or removal of specific deoxynucleotide triphosphate in the reaction mixture. Herein we report on the design of two novel assay protocols that utilize a dideoxynucleotide-terminated template strand and a phosphorothiolate-modified deoxynucleotide-terminated template strand. We designed and synthesized a deoxyuridine triphosphate analogue (dU*TP) containing 2-bromoisobutyryl group and demonstrated that it could be well recognized by φ29DNA polymerase, E. coli DNA polymerase I Klenow Fragment, Bst DNA polymerase Large Fragment, and E. coli DNA polymerase I Klenow Fragment (exo − ), which translated to effective incorporation of dU*TP into DNA. dU*TP was also successfully incorporated into extremely long single-stranded DNA at high-density using φ29 DNA polymerase by rolling circle amplification.