Laboratory of Foodomics, Institute of Food Science Research (CSIC), Madrid, Spain.
J Sep Sci. 2011 May;34(9):1011-9. doi: 10.1002/jssc.201000826. Epub 2011 Mar 15.
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%.
新的 DNA 扩增方法不断被开发出来，用于敏感检测和定量特定的 DNA 靶序列，例如临床、环境或食品应用。这些新的应用通常需要使用长 DNA 寡核苷酸作为靶序列杂交的探针。根据分子技术的不同，DNA 探针的长度从 40 到 450 个核苷酸不等，固相化学合成通常是生产这些探针的策略。然而，对于较大的 DNA 探针，DNA 化学合成的保真度会降低。寡核苷酸序列中的缺陷会导致杂交效率的丧失，从而影响扩增方法的灵敏度和选择性。在这项工作中，开发了一种酶促程序作为固相化学合成的替代方法，用于生产长寡核苷酸。探针生产的酶促程序基于短 DNA 序列的连接。长 DNA 探针是从小的寡核苷酸获得的，还有一小段序列作为桥，稳定用于 DNA 连接的分子复合物。通过毛细管凝胶电泳-激光诱导荧光检测（CGE-LIF）监测连接反应，使用裸熔融石英毛细管。毛细管凝胶电泳-LIF 方法非常有用和信息丰富，可用于表征连接反应，提供有关某些杂质性质的重要信息，以及精细优化连接条件（即连接循环、寡核苷酸和酶浓度）。结果，提高了连接产物的产量和质量。在实验室制备的 DNA 探针用于一种新的多重连接依赖性基因组扩增（MLGA）方法，用于检测样品中的转基因玉米。整个方法的巨大可能性通过特异性和敏感地检测低于 1%的转基因玉米得到了证明。
