Department of Chemistry, University of Alberta, Edmonton, AB T6G 2G2, Canada; Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Alexandria University, Alexandria, Egypt.
Department of Chemistry, University of Alberta, Edmonton, AB T6G 2G2, Canada.
Anal Chim Acta. 2017 Nov 22;994:92-99. doi: 10.1016/j.aca.2017.09.036. Epub 2017 Sep 28.
The human genome is susceptible to change; base mismatches can arise from damaged DNA, replication errors, and spontaneous mutation, and have the potential to cause apoptosis, carcinogenesis, and mutagenesis. Many techniques have been developed for DNA mismatch detection, but many of these methods have complex, time-consuming procedures and are limited to the detection of specific types of DNA mismatches. In this work, we present a general method for the simple and sensitive nucleobase-sensitized luminescent detection of mismatches in double-stranded DNA (dsDNA) using terbium ions. Terbium ions luminesce differently depending on the site of coordination in DNA due to the proximity effect of the energy transfer process that occurs from excited, non-hydrogen bonded nucleobases in single-stranded DNA (ssDNA) regions to the terbium ions. We examined the effect of location and number of mismatches on the sensitivity and selectivity of this probe in both synthetic oligonucleotides containing mismatches and natural calf thymus DNA exposed to UV light to induce reduced base pairing due to damage. This method shows good sensitivity for the determination of DNA mismatches, with limit of detection and limit of quantification of 1 and 3 mismatches, respectively, per dsDNA sequence.
人类基因组易发生变化；碱基错配可能源于受损的 DNA、复制错误和自发突变，并有导致细胞凋亡、致癌和致突变的潜力。已经开发出许多用于检测 DNA 错配的技术，但这些方法中的许多方法都具有复杂、耗时的程序，并且仅限于检测特定类型的 DNA 错配。在这项工作中，我们提出了一种使用铽离子简单、灵敏的核苷敏化荧光检测双链 DNA（dsDNA）中错配的通用方法。由于能量转移过程的临近效应，铽离子在 DNA 中的配位位置不同，其发光方式也不同，该过程发生在单链 DNA（ssDNA）区域中激发的、非氢键结合的核苷与铽离子之间。我们研究了位置和错配数量对该探针在含有错配的合成寡核苷酸和自然小牛胸腺 DNA 中的灵敏度和选择性的影响，小牛胸腺 DNA 暴露在紫外线下会因损伤而导致碱基配对减少。该方法对 DNA 错配的测定具有良好的灵敏度，对于每个 dsDNA 序列，检测限和定量限分别为 1 个和 3 个错配。
