Improved in situ hybridization efficiency with locked-nucleic-acid-incorporated DNA probes
Improved in situ hybridization efficiency with locked-nucleic-acid-incorporated DNA probes
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DOI：
10.1128/aem.03039-05
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发表时间：
2006-08-01
影响因子：
4.4
通讯作者：
Harada, Hideki
中科院分区：
文献类型：
--
作者：
Kubota, Kengo;Ohashi, Akiyoshi;Harada, Hideki
探针杂交效率低导致的信号强度低是rrna靶向原位杂交的主要缺陷之一。影响杂交效率的主要因素有两个：探针的可及性和对目标rRNA分子的亲和力。在这项研究中，我们证明了使用锁定核酸（LNA）结合的寡脱氧核苷酸探针（LNA/DNA探针）在不影响特异性的情况下显著提高了原位杂交效率。用2 ~ 4个LNA取代的荧光标记LNA/DNA探针，其荧光强度等于或大于Eub338探针，尽管这些探针在合成DNA探针时没有显示出明亮的信号；例如，用三个LNA碱基取代DNA碱基后，探针Eco468的荧光强度提高了22倍。探针的解离谱表明，解离温度与LNA取代数和荧光强度直接相关。这些结果表明，在DNA探针中引入LNA残基将是一种有效提高探针杂交效率的有效方法。
Low signal intensity due to poor probe hybridization efficiency is one of the major drawbacks of rRNA-targeted in situ hybridization. There are two major factors affecting the hybridization efficiency: probe accessibility and affinity to the targeted rRNA molecules. In this study, we demonstrate remarkable improvement in in situ hybridization efficiency by applying locked-nucleic-acid (LNA)-incorporated oligodeoxynucleotide probes (LNA/DNA probes) without compromising specificity. Fluorescently labeled LNA/DNA probes with two to four LNA substitutions exhibited strong fluorescence intensities equal to or greater than that of probe Eub338, although these probes did not show bright signals when they were synthesized as DNA probes; for example, the fluorescence intensity of probe Eco468 increased by 22-fold after three LNA bases were substituted for DNA bases. Dissociation profiles of the probes revealed that the dissociation temperature was directly related to the number of LNA substitutions and the fluorescence intensity. These results suggest that the introduction of LNA residues in DNA probes will be a useful approach for effectively enhancing probe hybridization efficiency.