High-Resolution Hydrodynamic Chromatographic Separation of Large DNA Using Narrow, Bare Open Capillaries: A Rapid and Economical Alternative Technology to Pulsed-Field Gel Electrophoresis? Lei Liu, † Vijaykumar Veerappan, ‡,⊥ Qiaosheng Pu, § Chang Cheng, ∥ Xiayan Wang,* ,† Liping Lu, † Randy D. Allen, ‡ and Guangsheng Guo* ,† † Department of Chemistry and Chemical Engineering, Beijing University of Technology, Beijing 100124, China ‡ Institute for Agricultural Biosciences, Oklahoma State University, Ardmore, Oklahoma 73401, United States § Department of Chemistry, Lanzhou University, Lanzhou, Gansu 730000, China ∥ Analytical Department, Albany Molecular Research, Inc., Rensselaer, New York 12144, United States * S Supporting Information ABSTRACT: A high-resolution, rapid, and economical hydro- dynamic chromatographic (HDC) method for large DNA separations in free solution was developed using narrow (5 μm diameter), bare open capillaries. Size-based separation was achieved in a chromatographic format with larger DNA molecules being eluting faster than smaller ones. Lambda DNA Mono Cut Mix was baseline-separated with the percentage resolutions generally less than 9.0% for all DNA fragments (1.5 to 48.5 kbp) tested in this work. High efficiencies were achieved for large DNA from this chromatographic technique, and the number of theoretical plates reached 3.6 × 10 5 plates for the longest (48.5 kbp) and 3.7 × 10 5 plates for the shortest (1.5 kbp) fragments. HDC parameters and performances were also discussed. The method was further applied for fractionating large DNA fragments from real-world samples (SacII digested Arabidopsis plant bacterial artificial chromosome (BAC) DNA and PmeI digested Rice BAC DNA) to demonstrate its feasibility for BAC DNA finger printing. Rapid separation of PmeI digested Rice BAC DNA covering from 0.44 to 119.041 kbp was achieved in less than 26 min. All DNA fragments of these samples were baseline separated in narrow bare open capillaries, while the smallest fragment (0.44 kbp) was missing in pulsed- field gel electrophoresis (PFGE) separation mode. It is demonstrated that narrow bare open capillary chromatography can realize a rapid separation for a wide size range of DNA mixtures that contain both small and large DNA fragments in a single run. M odern technologies for separating DNA fragments with high speed and high resolution is critical for the advancement of molecular biological and genomics research. High-performance liquid chromatography (HPLC) and electro- phoresis are the two primary techniques for DNA separations. Different HPLC methodologies, such as ion-pair reversed-phase LC, 1−3 size-exclusion chromatography, 4,5 slalom chromatog- raphy, 6−8 and hydrodynamic chromatography 9,10 have been employed, but the low resolving power of these methods prevents HPLC from competing with gel electrophoresis for DNA separation. As a result, agarose gel electrophoresis is most frequently used. However, conventional gel electrophoresis loses its efficiency for DNA molecules larger than ∼20 kilo base-pairs (kbp). 11 pulsed field gel electrophoresis (PFGE), which was developed in the early 1980s, is almost exclusively used for resolving large DNA molecules (≥10 kbp). 12,13 While it can achieve high resolutions for large DNA separations, PFGE is a tedious and time-consuming assay that requires large sample volumes. 14 These limitations led researchers to seek alternative methods for efficient separation of high molecular weight DNA samples. Recent advancements in micro/nano electromechanical systems have led to the fabrication of well- organized artificial structures to mimic sieving matrices in microchips to resolve DNA molecules. These arti ficial structures include entropic traps, 15,16 nanoslits, 17−19 nano- channels, 20−22 micro/nano pillars, 23,24 nanopores, 25−27 and other structures. 28−32 Although, these techniques provide faster analyses and reduced sample volumes, limited resolution continues to constrain the practical applications of these devices. Most recently, our group has made efforts to improve chromatographic DNA separations. We have developed a novel Received: October 4, 2013 Accepted: November 24, 2013 Published: November 24, 2013 Article pubs.acs.org/ac © 2013 American Chemical Society 729 dx.doi.org/10.1021/ac403190a | Anal. Chem. 2014, 86, 729−736