Characterization of visible dyes for four-decay fluorescence detection in DNA sequencing
Characterization of visible dyes for four-decay fluorescence detection in DNA sequencing
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DOI：
10.1021/ac961281p
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发表时间：
1997-07-01
影响因子：
7.4
通讯作者：
McGown, LB
中科院分区：
文献类型：
--
作者：
Nunnally, BK;He, H;McGown, LB
研究了几类染料作为用于DNA测序的多重四衰变荧光检测方案的候选物。染料包括硝基苯并呋喃染料、罗丹明染料、荧光素染料、花青染料、尼罗红和BODIPY染料。基于荧光光谱和寿命研究的结果，选择四种染料的初始组用于进一步研究：NBD-氨基己酸（NBD-HA，tau = 1.1 ns）、四甲基罗丹明甲酯（tau = 2.2 ns）、罗丹明绿色（tau = 4.3 ns）和BODIPY 505/515（tau = 5.9 ns）。四种染料的寿命检测的限制进行了研究，并证明了寿命分辨率的混合物中的游离染料在批处理溶液。染料标记的DNA引物的寿命也在批溶液中测定，并在毛细管电泳（CE）中检测。染料与DNA的结合提高了批量测量中混合物中染料各自寿命的分辨率。当连接到引物，四甲基罗丹明表现出双指数衰减的优势寿命为3.8纳秒，使其不适合四衰变测序。与CE凝胶接触延长了NBD-HA标记引物的寿命，从1.3到2.1 ns，但不影响其他染料的寿命。标记引物在电泳峰沿着各点的寿命可检测性在阿托摩尔范围内。
Dyes of several classes were investigated as candidates for use in a multiplex, four-decay fluorescence detection scheme for DNA sequencing. The dyes include nitrobenzofuran dyes, rhodamine dyes, fluorescein dyes, cyanine dyes, Nile Red, and BODIPY dyes. Based on the results of fluorescence spectral and lifetime studies, an initial set of four dyes was selected for further study: NBD-aminohexanoic acid (NBD-HA, tau = 1.1 ns), tetramethylrhodamine, methyl ester (tau = 2.2 ns), rhodamine green (tau = 4.3 ns), and BODIPY 505/515 (tau = 5.9 ns). Limits of lifetime detection of the four dyes were investigated, and lifetime resolution was demonstrated for mixtures of the free dyes in batch solution. Lifetimes of dye-labeled DNA primers also were determined in batch solution and detected on-the-ny in capillary electrophoresis (CE). Conjugation of the dyes to DNA improved the resolution of their individual lifetimes in mixtures in batch measurements. When attached to the primer, tetramethylrhodamine exhibited biexponential decay with a dominant lifetime of 3.8 ns, making it unsuitable for four-decay sequencing. Contact with the CE gel lengthened the lifetime of NBD-HA-labeled primer from 1.3 to 2.1 ns but did not affect the lifetimes of the other dyes. Lifetime detectability of labeled primers at individual points along an electrophoretic peak is in the attomole range.