Research Article High-fidelity fractionation of ssDNA fragments differing in size by one-base on a spiral-channel electrophoretic chip For the fractionation of fragments of interest from selective PCR products generated by high coverage gene expression profiling (HiCEP) analysis, high-resolution with the ability to discriminate and fractionate fragments differing by one base (base pair) in size is highly required. We report here on a new 4-inch diameter spiral-channel chip device for automatic high-fidelity fractionation. Overlapping DNA fragments of 180, 181 and 182 bases, with only one-base difference in size, were successfully fractionated. The collected fragments were PCR amplified, and then evaluated by size checking analysis, DNA sequencing, and homolog search. The high-resolution fractionation has been achieved because of the combined contributions of (i) the high-resolution separation using a 30 cm long spiral channel, (ii) a blocking technique to avoid contamination from unselected fragments during CE, and (iii) precise micro-scale target extraction. Contaminations due to unselected fractions have been greatly decreased to a negligible level by optimization of the extraction position and extraction time corresponding to the targeted segment only. This technique can be adapted to a wide range of applications, such as protein or cell collections where requirements for the high purity are more important than the amount of recovered fractionated material. Keywords: Contamination / Fraction collection / Fractionation / Electrophoresis / High coverage expression profiling / Spiral channel DOI 10.1002/elps.200900455 1 Introduction The Human Genome Project (HGP) has been followed by the comprehensive analyses of entire transcripts (transcriptome) to understand the molecular mechanisms underlying life [1]. Recently, the biological functions of non-coding transcripts have been defined as well, yet; the functions of many more remain to be discovered [2–5]. High coverage gene expression profiling (HiCEP), which is a cDNA-amplified restriction fragment length polymorphism (AFLP) based method, has been developed [6]. In contrast to hybridization procedures, the HiCEP analysis does not require any sequence informa- tion beforehand, allowing us to detect unknown transcripts and analyze any organism. However, there is considerable difficulty with a method using this AFLP principle, namely, it is necessary to collect fragments of interest and determine their sequences to know their original regions in the genome after the analysis. The conventional fractionation technique based on gel electrophoresis is time-consuming, lab inten- sive, and has been handicapped by insufficient resolution. Thus, the development of an automatic fractionation technology is an absolutely key issue for the broad use of these cDNA-AFLP based methods. The primary issues in realizing this fractionation include (i) high resolution of the separation as the prerequisite of fractionation, and (ii) non- contamination or reduced contamination in the fractionated fractions as the final goal of material recovery. Nowadays in biotechnology, electrophoretic separation of ssDNA with one-base resolution is routinely achieved for Sanger sequencing using slab gel, CE, or microchip [7]. However, for sequencing purpose, contaminations from pre- or post-bases are acceptable as long as a dominant peak shows the correct base. However, for the fractionation purpose, contaminations should be completely minimized and was a primary motivation of this study. CE has played a key role in sequencing as an efficient and fast technique. However, it has limitations in serving as a tool for fractionation due to its 1-D nature. The general extraction method when using CE is to measure the velocity of the fragments when passing through the detection window and to estimate their arrival time at the exit (the anode end) [8–10]. This limits a possibility to collect the Kai Sun 1 Nobuko Suzuki 2 Zheyu Li 1 Ryoko Araki 2 Kosei Ueno 1 Saulius Juodkazis 1 Masumi Abe 2 Sumihare Noji 3 Hiroaki Misawa 1 1 Research Institute for Electronic Science, Hokkaido University, Sapporo, Japan 2 National Institute of Radiological Sciences, Chiba, Japan 3 Institute of Engineering and Science, The University of Tokushima, Tokushima, Japan Received July 29, 2009 Revised September 7, 2009 Accepted September 11, 2009 Abbreviations: AFLP, amplified restriction fragment length polymorphism; HiCEP, high coverage gene expression profiling Correspondence: Professor Hiroaki Misawa, Research Institute for Electronic Science, Hokkaido University, Kita-21 Nishi-10, Kita-ku, Sapporo 001-0021, Japan E-mail: misawa@es.hokudai.ac.jp Fax: 181-11-706-9359 & 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com Electrophoresis 2009, 30, 4277–4284 4277