Method for detection of single-base mismatches using bimolecular beacons
Method for detection of single-base mismatches using bimolecular beacons
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DOI：
10.1021/ja012374d
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发表时间：
2002-03-20
影响因子：
15
通讯作者：
Lahiri, J
中科院分区：
文献类型：
--
作者：
Frutos, AG;Pal, S;Lahiri, J
本文描述了一种使用DNA微阵列检测单碱基错配的方法，该方法不需要标记样品（“靶”）DNA。该方法是基于破坏连接到固定的DNA链（“探针”）的荧光团和含有猝灭剂的序列之间的荧光能量转移（FRET），该序列除了在询问位点的人工错配（例如5-硝基吲哚、3-硝基吡咯或脱碱基位点）之外是互补的。由于FRET受体的置换和未标记探针的杂交是双分子的，术语“双分子信标”用于描述这种方法。错配的分析是基于完全匹配的DNA靶和含有单碱基错配的DNA靶杂交后FRET中破坏量的差异。使用这种方法和寡核苷酸模型系统，A/C单碱基错配被成功地区分在大于使用表面固定的分子信标所观察到的水平。歧视的量是依赖于身份的人工错配，更大的歧视，观察到5-硝基吲哚（一个“通用”的基地）比一个无碱基的网站。G/T错配，被认为是特别难以检测，也成功地区分时，淬灭序列含有5-硝基吲哚。
This paper describes a method for the detection of single-base mismatches using DNA microarrays in a format that does not require labeling of the sample (“target”) DNA. The method is based on disrupting fluorescence energy transfer (FRET) between a fluorophore attached to an immobilized DNA strand (“probe”) and a quencher-containing sequence that is complementary except for an artificial mismatch (e.g. 5-nitroindole, 3-nitropyrole, or abasic site) at the site of interrogation. As the displacement of the FRET acceptor and hybridization of the unlabeled probe are bimolecular, the term “bimolecular beacons” is used to describe this approach. The analysis of a mismatch was based on differences in the amount of disruption in FRET upon hybridization of perfectly matched DNA targets and those containing single-base mismatches. Using this method and an oligonucleotide model system, A/C single-base mismatches were successfully discriminated at levels greater than that observed using surface-immobilized molecular beacons. The amount of discrimination was dependent on the identity of the artificial mismatch; greater discrimination was observed with 5-nitroindole (a “universal” base) than with an abasic site. G/T mismatches, considered to be particularly difficult to detect, were also successfully discriminated when quencher sequences containing 5-nitroindole were used.