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Production of long DNA probes by ligation reaction by capillary gel electrophoresis with laser-induced fluorescence detection

AutorGarcía-Cañas, Virginia ; Mondello, Monica; Cifuentes, Alejandro
Fecha de publicación2010
CitaciónMSB 2010
ResumenMany novel DNA amplification methods are being developed for sensitive detection and quantification of specific DNA target sequences for clinical, environmental and food testing. These new molecular methods often require the use of long DNA oligonucleotides as probes for hybridization to the target sequences. Depending on the molecular technique, the length of DNA probes ranges from 40 to 450 nucleotides. Chemical synthesis is the general strategy adopted for oligonucleotide production. However, there is a decrease in the fidelity of chemical synthesis of DNA with distance from the first position at the 3’-end of each oligonucleotide. Defects in the oligonucleotide sequence result in the loss of hybridization efficiency of the DNA probe, affecting the sensitivity and selectivity of the amplification method. In this work, an enzymatic procedure has been investigated as alternative to solid-phase chemical synthesis for the production of long oligonucleotides. The enzymatic procedure for probe production was based on ligation of short DNA sequences. Each long probe was ligated together from two or more smaller oligonucleotides using short sequences that act as bridges stabilizing the molecular complex for DNA ligation. The ligation reactions were analyzed by capillary gel electrophoresis with laser-induced fluorescence detection (CGELIF) using a bare fused silica capillaries. The effect of some parameters on the separation and detection of ligation products, such as temperature of analysis and the pretreatment of the sample with formamide, was studied. The CGE-LIF method demonstrated to be very useful and informative for the characterization of the ligation reaction, as well as for the optimization of the ligation conditions, such as temperature and enzyme concentration, aimed at increasing the yield of ligation products. In-lab prepared DNA probes were used in a novel Multiplex Ligation-Dependent Genome Dependent Amplification (MLGA) method for the simultaneous detection of three genetically modified maize lines in maize samples. The quality of the produced probes was demonstrated by the specific and sensitive detection of four genomic DNA targets, namely, one reference and three sequences from three varieties of transgenic maize.
DescripciónResumen del póster presentado al 25th International Symposium on Microscale BioSeparations celebrado en Praga (Republica Checa) del 21 al 25 de marzo de 2010.
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