Combining ligation reaction and capillary gel electrophoresis to obtain reliable long DNA probes

Abstract

New DNA amplification methods are continuously developed for sensitive detection and quantification of specific DNA target sequences for, e.g. clinical, environmental or food applications. These new applications often require the use of long DNA oligonucleotides as probes for target sequences hybridization. Depending on the molecular technique, the length of DNA probes ranges from 40 to 450 nucleotides, solid-phase chemical synthesis being the strategy generally used for their production. However, the fidelity of chemical synthesis of DNA decreases for larger DNA probes. Defects in the oligonucleotide sequence result in the loss of hybridization efficiency, affecting the sensitivity and selectivity of the amplification method. In this work, an enzymatic procedure has been developed as an 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. Long DNA probes were obtained from smaller oligonucleotides together with a short sequence that acts as bridge stabilizing the molecular complex for DNA ligation. The ligation reactions were monitored by capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF) using a bare fused-silica capillary. The capillary gel electrophoresis-LIF method demonstrated to be very useful and informative for the characterization of the ligation reaction, providing important information about the nature of some impurities, as well as for the fine optimization of the ligation conditions (i.e. ligation cycles, oligonucleotide and enzyme concentration). As a result, the yield and quality of the ligation product were highly improved. The in-lab prepared DNA probes were used in a novel multiplex ligation-dependent genome amplification (MLGA) method for the detection of genetically modified maize in samples. The great possibilities of the whole approach were demonstrated by the specific and sensitive detection of transgenic maize at percentages lower than 1%.

Recent advances in the application of capillary electromigration methods for food analysis and Foodomics

“…During MLGA method development, defects in the oligonucleotide sequence result in the loss of hybridization and ligation efficiencies. These defects, produced typically during solid‐phase synthesis of long oligonucleotides, were overcome by developing an enzymatic procedure that was monitored by CGE‐LIF 85. By means of a ligation reaction, long DNA probes were obtained from smaller oligonucleotides.…”

Capillary Electrophoresis Based on Nucleic Acid Detection as Used in Food Analysis

“…The UP‐MPCR‐CE‐LIF assay requires only 0.1 ng of DNA; UP‐MPCR‐CE‐LIF is also a promising method of GMO detection (Zhang and others ). García‐Cañas and others (, ) developed a multiplex ligation‐dependent genome amplification (MLGA)‐CGE‐LIF assay utilizing short DNA sequences to synthesize long DNA probes. MLGA‐CGE‐LIF was combined with CGE‐LIF to detect transgenic maize of which the GM content was at a proportion of less than 1% (Figure ).…”

“…Scheme for MLGA‐CGE‐LIF (García‐Cañas and others ). (1) Short fragments are synthesized and hybridized to a bridge (vector) complementary to the central region of the selector probe.…”

Characterization and Study of Transgenic Cultivars by Capillary and Microchip Electrophoresis

“…The method was successfully applied for the simultaneous detection of GMOs in maize samples with LODs of 0.2% for GA21, 0.3% for MON863 and 0.1% for MON810. In a further work, the same group described an enzymatic procedure for the production of long DNA probes for their use in MLGA-CGE-LIF for the detection of GMOs in maize [ 26 ]. Oligonucleotides are generally obtained by chemical synthesis but this approach is inefficient producing long oligonucleotides leading in general to mixtures of shorter oligonucleotides [ 27 ].…”