Replaceable sieving polymers are the fundamental component for high-resolution nucleic acids separation in CE. The choice of polymer and its physical properties play significant roles in influencing separation performance. Recently, reversible addition fragmentation chain transfer (RAFT) polymerization has been shown to be a versatile polymerization technique capable of yielding well-defined polymers previously unattainable by conventional free-radical polymerization. In this study, a high molecular weight poly-(N,N-dimethylacrylamide) (PDMA) at 765000 gmol(-1) with a polydispersity index of 1.55 was successfully synthesized with the use of chain transfer agent2-propionic acidyl butyl trithiocarbonate in a multistep sequential RAFT polymerization approach. This study represents the first demonstration of RAFT polymerization for synthesizing polymers with the molecular weight range suitable for high-resolution DNA separation in sieving electrophoresis. Adjustment of pH in the reaction was found to be crucial for the successful RAFT polymerization of high molecular weight polymer as the buffered condition minimizes the effect of hydrolysis and aminolysis commonly associated with trithiocarbonate chain transfer agents. The separation efficiency of 2-propionic acidyl butyl trithiocarbonate PDMA was found to have marginally superior separation performance compared to a commercial PDMA formulation, POP-CAP, of similar molecular weight range.