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 recircularization using restriction and ligation, leading to a higher amplification yield and enabling the generation of circular products. However, C2CA is a multistep, nonisothermal 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 °C. Our C2CAplus method is simple, robust, and produces large quantities of product DNA that can be seen with the naked eye.

A comprehensive review of Microfluidic approaches in cell-free synthetic biology

“…One grand challenge that has emerged in cell-free research is the bottom-up development of a synthetic cell (Maerkl, 2023). Efforts towards this challenge include creation of DNA replication (Sakatani et al, 2018;Van Nies et al, 2018;Ueno et al, 2021;Grasemann et al, 2023), tRNA replication (Hibi et al, 2020), energy and metabolism (Berhanu et al, 2019;Miller et al, 2020), vesicle structures and membrane synthesis (Deshpande et al, 2016;Bhattacharya et al, 2019;Blanken et al, 2020), and communication systems (Li et al, 2019;Buddingh et al, 2020). We recently demonstrated continuous self-replication of essential proteins in the PURE system (Lavickova et al, 2020).…”

Self-Replicating DNA-Based Nanoassemblies

“…The cross-catalytic replication via RCA of complementary nanostructures based on nanojunctions, a complex multicrossover nanostructure, and circles , have also been reported, but all of these processes required separate steps and additional processing enzymes to generate copies of the complementary nanostructures, which could then assemble. More recently a one-pot multienzyme procedure was reported for circle amplification, but it is intrinsically limited to circle architectures …”