Sequence Dependent Mobility of Short Oligonucleotides in Free- Solution Electrophoresis

INTRODUCTION Since completion of the human genome project, many additional organisms have been sequenced and ~1.8 million human genetic Single Nucleotide Polymorphisms (SNPs) have been identified and documented by the SNP consortium, http://snp.cshl.org. While the technology workhorses for these efforts have been gel-based and capillary electrophoresis DNA sequencers, there is increased interest in the development of SNP technologies that utilize mass spectrometry (MS) for SNP analysis (e.g. http://www.sequenom.com). Thus, methods to amplify, manipulate and sequence DNA segments that are less than 20 to 30 bases in length and are compatible with MS systems are being developed. In free solution, polyelectrolytes that are much longer than their persistence length have an electrophoretic mobility that is independent of fragment length [1]. This characteristic makes it difficult to separate long DNA in free solution and is the reason that both gel and capillary electrophoresis systems use additional polymers to provide a molecular sieve that allows separation of these molecules based on length. Short DNA fragments, however, show an increase in mobility with length which becomes constant for lengths more than several hundred bases [2]. Sieving matrices are therefore unnecessary in separations used for genetic identification of short DNA segments. Use of fluorescence detection does, however, require the attachment of dye molecules that can affect the mobility. Thus, electrophoretic separations of oligonucleotide-dye complexes actually utilize a strategy known as end-labeled free solution electrophoresis or ELFSE, which has been shown to be quite useful for intermediate-length DNA fragments [3]. Another trend in electrophoresis technology development is the utilization of microfluidic channels on a chip. There are a number of practical advantages to using microfluidic chips in lieu of standard capillaries. First is size. The chips used in this experiment are only 1.6 cm wide and 9.5 cm long. Because of the size, the buffer and sample volumes are reduced and it is possible to place many sample flow paths on a single chip. As an example of this multiplexed approach, the research group of Mathies has developed a number of assays utilizing helped to pioneer chip-based capillary electrophoresis systems [4]. The use of small channels facilitates the application of higher field strengths yielding good separation performance. Since the channels on a microfluidic chip can be manufactured with dimensions smaller than those found on most standard capillaries, the surface to volume ratio is higher resulting in greater heat dissipation and less joule heating at high applied fields. The ability to apply higher field strengths results in separation completion on the order of minutes instead of hours. In the present work, free-solution electrophoresis of 15-mers of a single base and the corresponding, hybridized double strands are presented. This work was motivated by previous studies that showed multiple-peak separations for various multi-base ssDNA sequences. Sequence dependent conformation changes have been observed in other systems and our goal was to assess the effects of sequence on the formation of multiple peaks in high-field free-solution electrophoresis of short ssDNA.