C2CAplus: a one-pot isothermal circle-to-circle DNA amplification system

Abstract

Rolling circle amplification (RCA) is a widely used DNA amplification method that uses circular template DNA as input and produces multimeric, linear single or double stranded DNA. Circle-to-circle amplification (C2CA) has further expanded this method by implementing product re-circularization using restriction and ligation, leading to a higher amplification yield, and enabling the generation of circular products. However, C2CA is a multistep, non-isothermal method, requiring multiple fluid manipulations and thereby compromises several advantages of RCA. Here, we improved C2CA to implement a one-pot, single step, isothermal reaction at temperatures ranging from 25 to 37{degrees}C. Our C2CAplus method is simple, robust, and produces large quantities of product DNA that can be seen with the naked eye.