High-sensitive analysis of DNA fragments by capillary gel electrophoresis using transient isotachophoresis preconcentration and fluorescence detection.
J Chromatogr A
; 1216(17): 3602-5, 2009 Apr 24.
Article
in En
| MEDLINE
| ID: mdl-19211105
ABSTRACT
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.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
DNA
/
Electrophoresis, Capillary
/
Electrophoresis
/
Ethidium
/
Fluorescent Dyes
Type of study:
Diagnostic_studies
/
Evaluation_studies
/
Qualitative_research
Language:
En
Journal:
J Chromatogr A
Year:
2009
Document type:
Article
Affiliation country: