title: High-fidelity fractionation of ssDNA fragments differing in size by one-base on a spiral-channel electrophoretic chip.
abstract: 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.
authors.affiliation: Research Institute for Electronic Science, Hokkaido University, Sapporo, Japan.
authors.forename: Kai
authors.initials: K
authors.lastname: Sun
authors.forename: Nobuko
authors.initials: N
authors.lastname: Suzuki
authors.forename: Zheyu
authors.initials: Z
authors.lastname: Li
authors.forename: Ryoko
authors.initials: R
authors.lastname: Araki
authors.forename: Kosei
authors.initials: K
authors.lastname: Ueno
authors.forename: Saulius
authors.initials: S
authors.lastname: Juodkazis
authors.forename: Masumi
authors.initials: M
authors.lastname: Abe
authors.forename: Sumihare
authors.initials: S
authors.lastname: Noji
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Misawa
mesh_terms: D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D019075:Electrophoresis, Capillary / Q000295:instrumentation*; D020869:Gene Expression Profiling
chemical_list: D004277:DNA, Single-Stranded