He H, Nunnally B K, Li L C, McGown L B
Department of Chemistry, P. M. Gross Chemical Laboratory, Duke University, Durham, North Carolina 27708-0346, USA.
Anal Chem. 1998 Aug 15;70(16):3413-8. doi: 10.1021/ac980170i.
Mixtures of dye-labeled, M13-forward DNA primers were separated by capillary gel electrophoresis and detected on-the-fly, using fluorescence lifetime measurements, to evaluate four-decay detection for multiplex DNA sequencing. Three different four-dye systems were used, two that were excited at 488 nm and one that was excited at 514 nm. Each dye-labeled primer was identified on the basis of the lifetime of the conjugated dye using nonlinear least squares or the maximum entropy method to analyze the lifetime data. Overlapping electrophoretic peaks were generated by making multiple injections of mixtures of the dye-labeled primers. The overlapping peaks were resolved by fitting the data to two-, three- or four-component lifetime models used in nonlinear least-squares analysis in which each lifetime component was fixed to the predetermined lifetime of the corresponding dye-labeled primer. In two of the dye systems, the lifetimes of the four dye-labeled primers were sufficiently different to allow peak resolution. In the other dye system, addition of 10% DMSO to the run buffer changed the lifetime of one dye-labeled primer, allowing it to be resolved from another dye-labeled primer with similar lifetime.
通过毛细管凝胶电泳分离染料标记的M13正向DNA引物混合物，并使用荧光寿命测量实时检测，以评估多重DNA测序的四衰减检测。使用了三种不同的四染料系统，两种在488nm激发，一种在514nm激发。使用非线性最小二乘法或最大熵方法分析寿命数据，根据共轭染料的寿命识别每个染料标记的引物。通过多次注射染料标记引物的混合物产生重叠的电泳峰。通过将数据拟合到非线性最小二乘分析中使用的二、三或四组分寿命模型来解析重叠峰，其中每个寿命组分固定为相应染料标记引物的预定寿命。在两种染料系统中，四种染料标记引物的寿命差异足够大，足以实现峰分离。在另一种染料系统中，向运行缓冲液中添加10%二甲基亚砜改变了一种染料标记引物的寿命，使其能够与寿命相似的另一种染料标记引物分离。
