Simultaneous detection of genetically modified organisms by multiplex ligation-dependent genome amplification and capillary gel electrophoresis with laser-induced fluorescence.
In this work, an innovative method useful to simultaneously analyze multiple genetically modified organisms is described. The developed method consists in the combination of multiplex ligation-dependent genome dependent amplification (MLGA) with CGE and LIF detection using bare-fused silica capillaries. The MLGA process is based on oligonucleotide constructs, formed by a universal sequence (vector) and long specific oligonucleotides (selectors) that facilitate the circularization of specific DNA target regions. Subsequently, the circularized target sequences are simultaneously amplified with the same couple of primers and analyzed by CGE-LIF using a bare-fused silica capillary and a run electrolyte containing 2-hydroxyethyl cellulose acting as both sieving matrix and dynamic capillary coating. CGE-LIF is shown to be very useful and informative for optimizing MLGA parameters such as annealing temperature, number of ligation cycles, and selector probes concentration. We demonstrate the specificity of the method in detecting the presence of transgenic DNA in certified reference and raw commercial samples. The method developed is sensitive and allows the simultaneous detection in a single run of percentages of transgenic maize as low as 1% of GA21, 1% of MON863, and 1% of MON810 in maize samples with signal-to-noise ratios for the corresponding DNA peaks of 15, 12, and 26, respectively. These results demonstrate, to our knowledge for the first time, the great possibilities of MLGA techniques for genetically modified organisms analysis.
García-Cañas
Virginia
V
Department of Food Analysis, Institute of Industrial Fermentations (CSIC), Madrid, Spain.
Mondello
Monica
M
Cifuentes
Alejandro
A
D001483:Base Sequence; D017931:DNA Primers; D004270:DNA, Circular / Q000737:chemistry / Q000378:metabolism; D018744:DNA, Plant / Q000737:chemistry / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000379:methods*; D005504:Food Analysis / Q000379:methods*; D030841:Food, Genetically Modified; D008967:Molecular Biology; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D013050:Spectrometry, Fluorescence; D003313:Zea mays / Q000737:chemistry / Q000235:genetics
D017931:DNA Primers; D004270:DNA, Circular; D018744:DNA, Plant