High-sensitive analysis of DNA fragments by capillary gel electrophoresis using transient isotachophoresis preconcentration and fluorescence detection

Xu Zhongqi

Esumi Toshiaki

Ikuta Natsuki

Hirokawa Takeshi

Journal of Chromatography A

In this report aimed on further development of a high-sensitivity capillary gel electrophoresis (CCE) method for analysis of DNA fragments, we firstly explored online transient isotachophoresis (tITP) preconcentration combined with fluorescence detection (FD). The fluorescence signal (excitation: 488 nm; emission: 590 nm) was generated using the intercalating dye of ethidium bromide (EB). It was found when the leading electrolyte (LE) was injected behind the sample zone, such a special tITP mode has significant advantages to solve the bubble formation issue and to improve the analytical performance stability. Two standard DNA samples, a 50 bp DNA step ladder and the phi X174/HaeIII digest. were used to evaluate the qualitative and quantitative abilities of the tITP-FD approach. A highly diluted sample (10,000-fold in the water, e.g. the phi X174/HaeIII digest diluted from 500 mu g/ml to the 50 ng/ml level) was enriched and detected; the LOD was down to 0.09 ng/ml for the 72 bp fragment, apparently improved more than 1000-fold in comparison with UV detection. Although the RSD of peak areas (n = 3) was around 15.5% for the sample was electrokinetically injected, good linearity of peak area response showed that the proposed method is suitable for quantitative analysis.

Capillary gel electrophoresis

DNA fragments

Transient isotachophoresis

Fluorescence detection

Ethidium bromide

Chemical technology [ 570 ]

eng

Elsevier Science BV

Copyright (c) 2009 Elsevier B.V.

[ISSN] 0021-9673

[DOI] 10.1016/j.chroma.2009.01.066

[NCID] AA00695371

[DOI] http://dx.doi.org/10.1016/j.chroma.2009.01.066