Aqueous two-phase systems have frequently been utilized as an efficient bioseparation tool in the downstream processing of different biomolecules. The poly(ethyleneglycol)/poly(acrylate)/salt system, already explored successfully for purification of proteins and plasmid DNA, is here presented as an alternative approach for the isolation of small DNA fragments generated during in vitro DNA polymerase chain reactions. The polymerase chain reaction (PCR) is one of the most versatile laboratory techniques, but the purification of the amplified fragments often represents a major bottleneck. In this work we describe a simple and cost-effective method for isolation of DNA fragments obtained from PCR mixtures. The composition of the aqueous two-phase system in this work has been chosen to precipitate DNA molecules larger than 5000-7000 bp in the interphase, while the PCR products are partitioned very strongly between the phases in a two-step extraction process. In the first step, the DNA is partitioned quantitatively to the poly(ethyleneglycol)-phase. In the second, i.e. the back-extraction step, the DNA is strongly partitioned to a salt-rich phase that contains only low amounts of polymer. This system promotes rapid and high yields of purified small DNA products, less than 4000 bp, without contamination of proteins or large DNA templates present in the reaction mixture. (C) 2013 Elsevier B.V. All rights reserved.