High-sensitive analysis of DNA fragments by capillary gel electrophoresis using transient isotachophoresis preconcentration and fluorescence detection.
In this report aimed on further development of a high-sensitivity capillary gel electrophoresis (CGE) 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 phiX174/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 phiX174/HaeIII digest diluted from 500 microg/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.
Xu
Zhongqi
Z
Department of Applied Chemistry, Graduate School of Engineering, Hiroshima University, Higashi-hiroshima, Japan.
Esumi
Toshiaki
T
Ikuta
Natsuki
N
Hirokawa
Takeshi
T
D004247:DNA / Q000032:analysis* / Q000737:chemistry; D004586:Electrophoresis / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods*; D004996:Ethidium / Q000032:analysis*; D005456:Fluorescent Dyes / Q000032:analysis*; D007364:Intercalating Agents / Q000032:analysis; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D007364:Intercalating Agents; D004247:DNA; D004996:Ethidium