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.
Sun
Kai
K
Research Institute for Electronic Science, Hokkaido University, Sapporo, Japan.
Suzuki
Nobuko
N
Li
Zheyu
Z
Araki
Ryoko
R
Ueno
Kosei
K
Juodkazis
Saulius
S
Abe
Masumi
M
Noji
Sumihare
S
Misawa
Hiroaki
H
D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D019075:Electrophoresis, Capillary / Q000295:instrumentation*; D020869:Gene Expression Profiling
D004277:DNA, Single-Stranded