Please use this identifier to cite or link to this item: http://hdl.handle.net/1959.3/192817

Title

High-fidelity fractionation of ssDNA fragments differing in size by one-base on a spiral-channel electrophoretic chip

Author(s)

Sun, Kai;  Suzuki, Nobuko;  Li, Zheyu;  Araki, Ryoko;  Ueno, Kosei;  Juodkazis, Saulius;  Abe, Masumi;  Noji, Sumihare;  Misawa, Hiroaki

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.

Publication type

Journal article

Source

Electrophoresis, Vol. 30, no. 24 (Dec 2009), pp. 4277-4284

Publication year

2009

FOR Code(s)

0301 Analytical Chemistry;  0707 Veterinary Sciences

Keyword(s)

Contamination;  Fraction collection;  Fractionation;  Electrophoresis;  High coverage expression profiling;  Spiral channel

Publisher

Wiley-VCH Verlag

ISSN

0173-0835

Publisher URL

http://dx.doi.org/10.1002/elps.200900455

Copyright

Copyright © 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Additional information

The authors acknowledge support from the Japan Science and Technology Agency (Development of Systems and Technology for Advanced Measurement and Analysis Project), the Ministry of Education, Culture, Sports, Science, and Technology of Japan (KAKENHI Grant-in-Aid No. 19049001 for Scientific Research on the Priority Area 'Strong Photon-Molecule Coupling Fields for Chemical Reactions' No. 470), and Grantin-Aid from Hokkaido Innovation through Nanotechnology Support (HINTS).

Peer reviewed