FLUOROMETRIC ASSAY USING DIMERIC DYES FOR DOUBLE-STRANDED AND SINGLE-STRANDED-DNA AND RNA WITH PICOGRAM SENSITIVITY
FLUOROMETRIC ASSAY USING DIMERIC DYES FOR DOUBLE-STRANDED AND SINGLE-STRANDED-DNA AND RNA WITH PICOGRAM SENSITIVITY
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DOI：
10.1006/abio.1993.1020
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发表时间：
1993-01-01
影响因子：
2.9
通讯作者：
GLAZER, AN
中科院分区：
文献类型：
--
作者：
RYE, HS;DABORA, JM;GLAZER, AN
噻唑橙同二聚体（TOTO；1,1'-（4,4,7,7-四甲基-4,7-二氮十一亚甲基）-双-4-[3-甲基-2,3-二氢-（苯并-1,3-噻唑）-2-亚甲基]-四碘化喹啉）和恶唑黄同二聚体（YOYO；TOTO 的类似物，用苯并-1,3-恶唑代替苯并-1,3-噻唑）以非常高的亲和力与核酸结合，结合后荧光增强超过 1000 倍。在 4 mM Tris-acetate/0.1 mM EDTA/50 mM NaCl，pH 8.2 中存在 2 × 10−7M TOTO 或 YOYO 的情况下，荧光强度与 DNA 浓度在 0.5 至 100 ng/ml 范围内呈线性关系，允许在传统荧光计中对双链 DNA 进行灵敏定量。将核酸-染料混合物置于低自发荧光塑料块中的 25 µl 孔阵列中，并使用激光激发共聚焦荧光扫描仪进行检测，每孔可检测到少至 20 pg 的双链 DNA。阵列扫描法快速、通量高、样品用量少。它还可以对单链 DNA 和 RNA 进行定量。
Thiazole orange homodimer (TOTO; 1,1′-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)-bis-4-[3-methyl-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene]-quinolinium tetraiodide) and oxazole yellow homodimer (YOYO; an analogue of TOTO with a benzo-1,3-oxazole in place of the benzo-1,3-thiazole) bind with very high affinity to nucleic acids with more than a 1000-fold fluorescence enhancement upon binding. A linear dependence of fluorescence intensity on DNA concentration over a range from 0.5 to 100 ng/ml in the presence of 2 × 10−7M TOTO or YOYO in 4 mM Tris-acetate/0.1 mM EDTA/50 mM NaCl, pH 8.2 allows sensitive quantitation of double-stranded DNA in a conventional fluorometer. With nucleic acid-dye mixtures in an array of 25-μl wells in a block of low autofluorescence plastic and detection with a laser-excited confocal fluorescence scanner, as little as 20 pg of double-stranded DNA can be detected per well. The array scanning method is rapid, has high throughput, and requires small amounts of sample. It also allows quantitation of single-stranded DNA and RNA.