Ming-Mu Hsieh Po-Ling Chang Huan-Tsung Chang Department of Chemistry, National Taiwan University, Taipei, Taiwan, R.O.C. The impact of a plug of salts on the analysis of large volumes of dsDNA by capillary electrophoresis A partially filling technique for the analysis of DNA markers and polymerase chain reac- tion (PCR) products by capillary electrophoresis in the presence of electroosmotic flow using polymer solutions is presented. Either after or prior to the sample injection, a plug of salts at high pH was hydrodynamically injected. During the separation, poly(ethylene oxide) (PEO) solution entered the capillary. We have found that the position, length, and composition of the plugs affect the sensitivity, resolution, and speed on the analysis of FX-174/HaeIII DNA restriction fragments or a DNA mixture (pBR 322/HaeIII digest, pBR 328/BglI digest and pBR 328/HinfI digest) with different degrees. Through careful evaluation of the impact of anions and cations on the analysis of DNA, we have sug- gested that the optimal condition is applying a plug consisting of 32 mM NaCl and 0.01 M NaOH at 30 cm height for 60 s after sample injection. In the presence of such a plug, PEO adsorption reduces, and thus the separation is faster, as well as the sensi- tivity improves. Using this condition, the analysis of a DNA mixture (injected at 30 cm for 360 s) containing ten different PCR products amplified after 17 cycles was complete in 25 min. About a 2000-fold improvement in the sensitivity was achieved when com- pared to that by a conventional method (10 s injection) without applying a plug. Keywords: Capillary electrophoresis / DNA / Laser-induced fluorescence / Polymer solutions EL 5051 1 Introduction Owing to their ease of use and replacement, many hydro- philic polymer solutions like poly(ethylene oxide) (PEO) have been tested in capillary electrophoresis (CE) for the analysis of polymerase chain reaction (PCR) products and DNA sequencing for many years with varying degrees of success [1–11]. Despite the sieving ability of the polymer solution, the use of coated capillary to suppress electro- osmotic flow (EOF) and to minimize DNA adsorption is generally required for high resolution and reproducibility [12–15]. Recently, we have demonstrated the analysis of DNA using PEO in the presence of EOF [16–21]. After injection of DNA, PEO enters the capillary filled with tris(hydroxymethyl)aminomethane (Tris) and borate (TB) buffer. Because DNA migrates against EOF, large DNA is detected earlier towards the cathodic end. Through a ser- ies of studies, we have learned that PEO adsorption on the capillary wall is easily removed by washing with 0.5 M NaOH at 1 kV for 10 min after each run. In order to control PEO adsorption and thus regulate EOF, the ionic strength and pH of the buffers used to fill the capillary and prepare polymer solutions are of considerable importance [17– 20], especially, when on-line concentration and separa- tion of DNA in the presence of EOF using PEO was per- formed [22]. In order to optimize DNA separation by CE, additives such as amines, metal ions, and urea have been added to background electrolytes [23–26]. This is because such additives could cause changes with different degrees in DNA conformation and charge density, the binding con- stant for intercalated DNA complex, viscosity, as well as the property of the capillary wall. For instance, it has been found that DNA separation is better in high-pH buffer con- taining NaCl than in conventional Tris-borate-EDTA (TBE) buffer [27], mainly due to a small diffusion coefficient of DNA. The increment in the mobility with increasing the concentration of alkaline metal ions and the improvement in the efficiency for the DNA separation in the presence of lithium ions have recently been reported [26]. However, the binding between DNA and ethidium bromide (EtBr) decreases with increasing the concentration of salts, leading to decreases in the sensitivity [28]. Although the separation of DNA using solutions containing suitable amounts of salts at high pH is quite promising, a particular attention must be paid to minimize Joule heating. To achieve this goal, our attempt was to develop a partially filling technique for DNA separation in the presence of EOF using PEO solutions. The effects of a plug of salts Correspondence: Dr. Huan-Tsung Chang, Department of Chem- istry, National Taiwan University, Section 4, Rooseve.lt Road, Tai- pei, Taiwan, R.O.C. E-mail: changht@ccms.ntu.edu.tw Fax: 1886-2-23621963 Abbreviations: EtBr , ethidium bromide; PEO, poly(ethylene oxide); TB, Tris-borate 2388 Electrophoresis 2002, 23, 2388–2393  WILEY-VCH Verlag GmbH, 69451 Weinheim, 2002 0173-0835/02/1508–2388 $17.501.50/0