AbstractAn efficient PCR amplification of various templates (short 57‐mer, random 67‐ and 82‐mer, and long DNA) with base‐modified nucleoside triphosphates is presented here. Using 5‐substituted pyrimidine and 7‐substituted‐7‐deaza‐ or 8‐substituted purine nucleoside triphosphates as substrates for thermostable DNA polymerases [Taq and Vent (exo–)], successful PCR amplification of partially or entirely modified DNA libraries and long DNA constructs (up to 1.5 kb) is achieved. Visualization of double‐stranded PCR product formation is improved through the use of primers with different fluorescent labels. This allows one to monitor the efficiency of modified substrate incorporation and the enzymatic recognition of the modified template during PCR. The redesigned fully base‐modified DNA (denoted ‘DZA’) can be utilized for the straightforward production of diverse libraries for in vitro selection of aptamer and catalytic nucleic acids as well as for the synthesis of artificial genetic templates, replicons, or complex vectors. © 2018 by John Wiley & Sons, Inc.