Isothermal nucleic acid amplification techniques are attracting increasing attention in molecular diagnosis and biotechnology. However, most existing techniques are complicated by the need for intricate primer design and numerous enzymes and primers. Here, we have developed a simple method, termed NAQ, that employs adding both endonuclease Q (EndoQ) and dUTP/dITP to conventional rolling circle amplification reactions to increase DNA amplification. NAQ does not require intricate primer design or DNA sequence-specific enzymes, and existing isothermal amplification techniques could be readily adapted to include both EndoQ and dUTP/dITP. Display omitted •Endonuclease Q and dUTP/dITP improves rolling circle amplification (RCA).•Endonuclease Q is the optimal nickase for the improved reaction.•No intricate primer design or restriction enzymes are required.