Highly reproducible capillary gel electrophoresis (CGE) of DNA fragments using uncoated columns. Detection of genetically modified maize by PCR-cGE
✍ Scribed by Virginia García-Cañas; Ramón González; Alejandro Cifuentes
- Publisher
- John Wiley and Sons
- Year
- 2002
- Tongue
- English
- Weight
- 184 KB
- Volume
- 25
- Category
- Article
- ISSN
- 1615-9306
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✦ Synopsis
Highly reproducible capillary gel electrophoresis (CGE) of DNA fragments using uncoated columns.
Detection of genetically modified maize by PCR-CGE
In this work a capillary gel electrophoresis (CGE) method is presented that yields reproducible separations of DNA fragments using commercially available polymers together with bare fused silica capillaries. The method combines a washing routine of the column with 0.1 M hydrochloride acid followed by a rinsing step with a solution containing 1% polyvinyl alcohol. The use of this procedure together with a running Tris-phosphate-EDTA buffer containing 2-hydroxyethyl cellulose (HEC) at pH 7.3 gives highly resolved separations of DNA fragments ranging from 80 to 500 bp. The separation of these DNA fragments is achieved in ca. 20 min with efficiencies up to 1.8610 6 plates/m. Reproducibility values of migration times (given as %RSD) of the DNA fragments separated by this CGE method are better than 0.86% (n = 10) for the same day, 1.61% (n = 40) for four different days, and 1.4% (n = 15) for three different capillaries. The length up to 500 bp corresponds to the DNA sizes more frequently amplified by PCR for detecting genetically modified organisms (GMOs) in foods. The usefulness of this separation method is demonstrated by detecting genetically modified insect-resistant Bt maize after amplification of a DNA fragment by PCR. Detection of 1% of Bt maize in flour is carried out using this CGE procedure with UV absorbance and laser induced fluorescence (LIF).