Capillary Electrophoresis with Nanoparticle Matrix for DNA Analysis

Abstract

Biopolymer such as DNA is important in living cells since its genetic information provides many building blocks for cell components. Also, DNA is employed for diagnosis of genetic disease, DNA sequencing, forensic investigation, and post-human genome project. Conventional methods such as slab gel electrophoresis (SGE) and ion chromatography (IC) for DNA analysis suffered long separation time and low resolution. However, recently developed capillary gel electrophoresis (CGE) showed high speed and high separation efficiency for especially DNA separation. Capillary electrophoresis (CE) without sieving medium does not have enough separation power for DNA sample with different lengths since each DNA has very similar charge-to-mass ratio, which deteriorates mobility differences for various sizes of DNA under electric field. Therefore, development of the sieving medium is the key for efficient DNA analysis. In early era for the capillary format, agarose or crosslinked polyacrylamide had been used at the expense of bubble formation at high electric field, gel instability, and no room for gel replacement. Later, water-soluble polymers such as cellulose derivatives including methyl cellulose (MC), hydroxyethyl cellulose (HEC), and hydroxypropylmethyl cellulose (HPMC) had been employed for relatively short DNA fragment analysis. These polymers exhibited strong stability under high electric field and ease of replacement. Liner type polymers such as linear polyacrylamide (LPA), polyethylene oxide (PEO), polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA) also provided similar effects on DNA separation. However, either cellulose or linear type polymers had relatively high solution viscosity for high resolution of DNA samples, which made it difficult for automation and fast analysis. In high throughput analysis for DNA sequencing, single nucleotide polymorphism (SNP), and polymerase chain reaction (PCR) products, effective but low viscosity sieving medium is essential for high sieving ability and easy replacement. Recently, mannitol added poly(N-isopropylacrylamide) (PNIPAM) or gold nanoparticle (GNP) as the sieving medium showed the potential for this purpose. In GNP case, the production of GNP took long time and the cost was high for preparation. In this paper, we have employed relatively cheap silica or α-alumina nanoparticle as the sieving medium. These nanoparticles were successfully applied for DNA analysis with the size range of 500 bp to 5000 bp. Figure 1 shows the electropherograms of 500 bp step ladder by capillary electrophoresis with different sieving matrices. Since the direction of the electosmtic flow (EOF) caused by surface silanol groups on fused silica capillary is toward negative electrode (opposite compared to migration of DNA) and the mangnitude of EOF is usually larger than that of electrophoretic mobility of DNA, the capillary surface needs to be covered in order to reduce EOF. The capillary surface in this experiment was coated either permanently (Figure 1(a) and (b)) or dynamically (Figure 1(c) to (e)). Figure 1(a) exhibited the significance of sieving