Combining ligation reaction and capillary gel electrophoresis to obtain reliable long DNA probes
9 páginas, 6 figuras, 1 tabla.-- El pdf del artículo es la versión revisada de autor.
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Wiley-VCH
2011-05
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| Subjects: | CGE-LIF, DNA amplification, DNA ligase, Food analysis, |
| Online Access: | http://hdl.handle.net/10261/49782 http://dx.doi.org/10.13039/501100004837 |
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dig-cial-es-10261-497822018-08-23T10:50:25Z Combining ligation reaction and capillary gel electrophoresis to obtain reliable long DNA probes García-Cañas, Virginia Mondello, Monica Cifuentes, Alejandro Ministerio de Ciencia e Innovación (España) Ministerio de Educación y Ciencia (España) CGE-LIF DNA amplification DNA ligase Food analysis 9 páginas, 6 figuras, 1 tabla.-- El pdf del artículo es la versión revisada de autor. 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%. This work was supported by AGL2008-05108-C03-01 and 200970I083 (Ministerio de Ciencia e Innovación), and CSD2007-00063 FUN-C-FOOD (Programa CONSOLIDER, Ministerio de Educacion y Ciencia) projects. Peer reviewed 2012-05-17T07:49:21Z 2012-05-17T07:49:21Z 2011-05 artículo http://purl.org/coar/resource_type/c_6501 Journal of Separation Science 34(9): 1011-1019 (2011) 1615-9306 http://hdl.handle.net/10261/49782 10.1002/jssc.201000826 1615-9314 http://dx.doi.org/10.13039/501100004837 en http://dx.doi.org/10.1002/jssc.201000826 open Wiley-VCH |
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CGE-LIF DNA amplification DNA ligase Food analysis CGE-LIF DNA amplification DNA ligase Food analysis |
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CGE-LIF DNA amplification DNA ligase Food analysis CGE-LIF DNA amplification DNA ligase Food analysis García-Cañas, Virginia Mondello, Monica Cifuentes, Alejandro Combining ligation reaction and capillary gel electrophoresis to obtain reliable long DNA probes |
| description |
9 páginas, 6 figuras, 1 tabla.-- El pdf del artículo es la versión revisada de autor. |
| author2 |
Ministerio de Ciencia e Innovación (España) |
| author_facet |
Ministerio de Ciencia e Innovación (España) García-Cañas, Virginia Mondello, Monica Cifuentes, Alejandro |
| format |
artículo |
| topic_facet |
CGE-LIF DNA amplification DNA ligase Food analysis |
| author |
García-Cañas, Virginia Mondello, Monica Cifuentes, Alejandro |
| author_sort |
García-Cañas, Virginia |
| title |
Combining ligation reaction and capillary gel electrophoresis to obtain reliable long DNA probes |
| title_short |
Combining ligation reaction and capillary gel electrophoresis to obtain reliable long DNA probes |
| title_full |
Combining ligation reaction and capillary gel electrophoresis to obtain reliable long DNA probes |
| title_fullStr |
Combining ligation reaction and capillary gel electrophoresis to obtain reliable long DNA probes |
| title_full_unstemmed |
Combining ligation reaction and capillary gel electrophoresis to obtain reliable long DNA probes |
| title_sort |
combining ligation reaction and capillary gel electrophoresis to obtain reliable long dna probes |
| publisher |
Wiley-VCH |
| publishDate |
2011-05 |
| url |
http://hdl.handle.net/10261/49782 http://dx.doi.org/10.13039/501100004837 |
| work_keys_str_mv |
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