High-sensitivity two-color detection of double-stranded DNA with a confocal fluorescence gel scanner using ethidium homodimer and thiazole orange.
High-sensitivity two-color detection of double-stranded DNA with a confocal fluorescence gel scanner using ethidium homodimer and thiazole orange.
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DOI：
10.1093/nar/19.2.327
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发表时间：
1991-01
影响因子：
14.9
通讯作者：
H. Rye;M. Quesada;K. Peck;R. Mathies;A. Glazer
中科院分区：
文献类型：
--
作者：
H. Rye;M. Quesada;K. Peck;R. Mathies;A. Glazer
乙锭同二聚体（EthD；λ Fmax 620 nm）在 EthD:DNA 比例高达 1 染料:4-5 bp 时与双链 DNA (dsDNA) 形成稳定的荧光复合物，可使用共焦荧光凝胶扫描仪进行高灵敏度检测（Glazer, A.N., Peck, K. & Mathies, R.A. (1990) Proc. Natl. 阿卡德。科学。美国 87, 3851-3855)。然而，在与未标记的 DNA 一起孵育时，EthD 会从其与 dsDNA 的复合物部分迁移到未标记的 DNA。这里显示，这种迁移取决于原始 dsDNA-EthD 复合物中嵌入位点的占据分数，并且没有可检测到的来自 50 bp:1 染料形成的 dsDNA-EthD 复合物的转移。单嵌入剂噻唑橙（TO；λ Fmax 530 nm）与 dsDNA 形成易于解离的复合物，结合时荧光增强（Lee, L.G., Chen, C. & Liu, L.A. (1986) Cytometry 7, 508-517）。然而，大摩尔过量的 TO 并不能取代 EthD 与 dsDNA 的复合物。当 TO 和 EthD 结合到同一个 dsDNA 分子时，TO 的激发会导致从 TO 到 EthD 的有效能量转移。该观察结果表明使用具有非常高的吸收系数和有效的能量转移特性的第二嵌入染料“敏化”EthD 荧光的实用性。将预先形成的线性化 M13mp18 DNA-EthD 复合物与未标记的线性化 pBR322 一起在缓冲液中含有 TO 的琼脂糖凝胶上进行电泳，可以在 530 nm 处的 pBR322 DNA-TO 复合物和 620 nm 处的 M13mp18 DNA-EthD 复合物的同一泳道中进行灵敏的荧光检测。这些观察结果为在染色体物理作图等双色应用中使用携带数百个发色团的稳定 DNA 染料嵌入复合物奠定了基础。
Ethidium homodimer (EthD; lambda Fmax 620 nm) at EthD:DNA ratios up to 1 dye:4-5 bp forms stable fluorescent complexes with double-stranded DNA (dsDNA) which can be detected with high sensitivity using a confocal fluorescence gel scanner (Glazer, A.N., Peck, K. & Mathies, R.A. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3851-3855). However, on incubation with unlabeled DNA partial migration of EthD takes place from its complex with dsDNA to the unlabeled DNA. It is shown here that this migration is dependent on the fractional occupancy of intercalating sites in the original dsDNA-EthD complex and that there is no detectable transfer from dsDNA-EthD complexes formed at 50 bp: 1 dye. The monointercalator thiazole orange (TO; lambda Fmax 530 nm) forms readily dissociable complexes with dsDNA with a large fluorescence enhancement on binding (Lee, L.G., Chen, C. & Liu, L.A. (1986) Cytometry 7, 508-517). However, a large molar excess of TO does not displace EthD from its complex with dsDNA. When TO and EthD are bound to the same dsDNA molecule, excitation of TO leads to efficient energy transfer from TO to EthD. This observation shows the practicability of 'sensitizing' EthD fluorescence with a second intercalating dye having a very high absorption coefficient and efficient energy transfer characteristics. Electrophoresis on agarose gels, with TO in the buffer, of preformed linearized M13mp18 DNA-EthD complex together with unlabeled linearized pBR322 permits sensitive fluorescence detection in the same lane of pBR322 DNA-TO complex at 530 nm and of M13mp18 DNA-EthD complex at 620 nm. These observations lay the groundwork for the use of stable DNA-dye intercalation complexes carrying hundreds of chromophores in two-color applications such as the physical mapping of chromosomes.