HIGH-SENSITIVITY CAPILLARY ELECTROPHORESIS OF DOUBLE-STRANDED DNA FRAGMENTS USING MONOMERIC AND DIMERIC FLUORESCENT INTERCALATING DYES
HIGH-SENSITIVITY CAPILLARY ELECTROPHORESIS OF DOUBLE-STRANDED DNA FRAGMENTS USING MONOMERIC AND DIMERIC FLUORESCENT INTERCALATING DYES
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DOI：
10.1021/ac00085a004
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发表时间：
1994-07-01
影响因子：
7.4
通讯作者：
MATHIES, RA
中科院分区：
文献类型：
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作者：
ZHU, HP;CLARK, SM;MATHIES, RA
荧光检测毛细管电泳分离的phi X174/HaeIII DNA限制性内切片段进行了单体和二聚体插入染料。采用可替代羟乙基纤维素溶液作为分离介质。在488 nm激光激发下进行共聚焦荧光检测。测定了单体染料（溴化乙啶、两种丙啶染料衍生物、恶唑黄、噻唑橙和一种聚阳离子噻唑橙衍生物）柱上染色的DNA检出限。噻唑橙染料提供了最灵敏的检测，限制灵敏度为每带2-4个amol的DNA碱基对，并且成功地检测了603-bp的片段，从phi X174/HaeIII样品中注射，每微升仅含有1-2 fg的该片段。预先形成的dna -二聚体染料络合物的分离也进行了。在预先形成的DNA-二聚体染料复合物的分离中观察到的条带宽度是由于具有不同数量的结合染料分子的DNA片段的存在，这些分子在我们的许多分离中可以被分解为紧密间隔的亚带。用9-氨基吖啶（9AA）在色谱柱和运行缓冲液中进行电泳，可以显著提高这些dna -染料复合物分离的质量。二聚体染料TOTO、EthD、TOTAB和YOYO制备的配合物的最佳分离浓度分别为100 μ M、1 μ M、1 μ M和0.5 μ M。在这些9AA浓度下，分离是可重复的，它们具有优异的分辨率，并且可以从TOTO每微升稀释40 fg的DNA溶液中检测到603-bp的phi X片段，类似于YOYO和TOTAB每微升稀释100 fg， EthD每微升稀释200 fg。本工作为毛细管电泳双链DNA片段的常规高灵敏度荧光检测建立了条件。可靠地进行稳定的非共价dna -染料络合物的高质量分离的能力也将在多重检测方法的发展中有价值。
Fluorescence-detected capillary electrophoresis separations of phi X174/HaeIII DNA restriction fragments have been performed using monomeric and dimeric intercalating dyes. Replaceable hydroxyethyl cellulose solutions were used as the separation medium. Confocal fluorescence detection was performed following 488-nm laser excitation. The limits of DNA detection for on-column staining with monomeric dyes (ethidium bromide, two propidium dye derivatives, oxazole yellow, thiazole orange, and a polycationic thiazole orange derivative) were determined. The thiazole orange dyes provide the most sensitive detection with limiting sensitivities of 2-4 amol of DNA base pairs per band, and detection of the 603-bp fragment was successful, injecting from phi X174/HaeIII samples containing only 1-2 fg of this fragment per microliter. Separations of preformed DNA-dimeric dye complexes were also performed. The breadth of the bands observed in separations of preformed DNA-dimeric dye complexes is due to the presence of DNA fragments with different numbers of bound dye molecules that can be resolved as closely spaced subbands in many of our separations. The quality of these DNA-dye complex separations can be dramatically improved by performing the electrophoresis with 9-aminoacridine (9AA) in the column and running buffers. The optimum concentrations of 9AA for the separation of complexes preformed with the dimeric dyes TOTO, EthD, TOTAB, and YOYO were determined to be 100, 1, 1, and 0.5 mu M, respectively. At these 9AA concentrations, the separations are reproducible, they exhibit excellent resolution, and the 603-bp phi X fragment can be detected following injection from DNA solutions as dilute as 40 fg of this DNA fragment per microliter with TOTO, similar to 100 fg/mu L with YOYO and TOTAB, and 200 fg/mu L with EthD. This work establishes conditions for routine high-sensitivity fluorescence detection of double-stranded DNA fragments in capillary electrophoresis. The ability to reliably perform high-quality separations of stable noncovalent DNA-dye complexes will also be valuable in the development of multiplex detection methods.