Ultrasensitive detection of genetically modified maize DNA by capillary gel electrophoresis with laser-induced fluorescence using different fluorescent intercalating dyes.
In this work, four different fluorescent intercalating dyes are compared for the ultrasensitive CGE-LIF detection of DNA from transgenic maize in flours. The fluorescent intercalating dyes compared are YOPRO-1, SYBR-Green-I, Ethidium bromide (EthBr), and EnhanCE. For all the four dyes optimum concentrations are established, and efficient separations of DNA fragments ranging in size from 80 to 1000 bp are obtained. The comparative study demonstrates that SYBR-Green-I and YOPRO-1 provide better limits of detection (LODs) than EnhanCE or EthBr (i.e., LODs are, respectively, 700, 1000, 11300, and 97400 zmol, calculated for a 200-bp DNA fragment). Separations using YOPRO-1 are faster than those using SYBR-Green-I (30 min vs 47 min for the analysis of the 80-1000 bp DNA fragments). Also, separations using YOPRO-1 are more efficient than those using SYBR-Green-I (e.g., 2.4 x 10(6) plates/m vs 1.6 x 10(6) plates/m, respectively, calculated for the 200-bp fragment). Also, buffer depletion and cost per analysis are worse with SYBR-Green-I than with YOPRO-1. Therefore, YOPRO-1 was selected as the preferred intercalating dye. Using this fluorescent compound, analysis time reproducibility for the CGE-LIF separation of the DNA fragments is determined to be better than 1.7% (% RSD, n = 10) within the same day, and better than 1.9% (% RSD, n = 30) for three different days. Moreover, the fluorescence signal obtained using this dye is shown to vary linearly with the DNA concentration in the range studied, i.e., 1-500 ng/microL. It is demonstrated that by using this method 0.01% of transgenic maize can be detected in flour by direct injection of the PCR-amplified sample.
Institute of Industrial Fermentations (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain.
Virginia
V
García-Cañas
Ramón
R
González
Alejandro
A
Cifuentes
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics; D001427:Bacterial Toxins; D052160:Benzothiazoles; D003365:Costs and Cost Analysis; D018744:DNA, Plant / Q000032:analysis* / Q000302:isolation & purification; D003959:Diamines; D019075:Electrophoresis, Capillary / Q000379:methods*; D004731:Endotoxins / Q000235:genetics; D004996:Ethidium; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000191:economics; D006460:Hemolysin Proteins; D007364:Intercalating Agents; D007834:Lasers; D009930:Organic Chemicals; D030821:Plants, Genetically Modified; D016133:Polymerase Chain Reaction; D011804:Quinolines; D015203:Reproducibility of Results; D013214:Starch Synthase / Q000235:genetics; D003313:Zea mays / Q000235:genetics*
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D052160:Benzothiazoles; D018744:DNA, Plant; D003959:Diamines; D004731:Endotoxins; D005456:Fluorescent Dyes; D006460:Hemolysin Proteins; D007364:Intercalating Agents; D009930:Organic Chemicals; D011804:Quinolines; C030956:insecticidal crystal protein, Bacillus Thuringiensis; C098022:SYBR Green I; D013214:Starch Synthase; D004996:Ethidium