GENOME SEQUENCING BY FOUR DECAY FLUORESCENCE DETECTION

描述（改编自研究者摘要）： 荧光标记DNA的检测 用于基因组测序的片段正在研究中。 在这个多功能厅 方案，荧光寿命取代光谱选择性， 区分四个碱基特异性标签。 因此，该方案是 基于检测到四种荧光衰减而不是四种颜色， DNA片段的单道分离。 荧光寿命是 提供“数字”检测的浓度无关特性， 提供了优于模拟的、强度相关的测量的重要优点。 由于四种染料的荧光寿命是预先确定的，因此数据 当有不完整的情况时，分析是简单的，甚至是直接的 电泳分辨率 连续、实时荧光寿命 在频域中执行四衰减测序中的检测 使用最先进的商业仪器， 多谐波傅里叶变换（MHF）技术，允许测量 在许多调制频率下同时进行相位和调制。 美克美家 仪器与商业毛细管电泳（CE） 系统 荧光寿命和强度同时来自于 同样的测量，以产生一个寿命强度电泳图在没有 比简单的强度或彩色电泳图所需的时间更长。 事实上，提高精度和分辨率，这是预期从生命周期 检测应该能够实现更快的测序，因为 电泳分辨率将变得容易。 下一个项目的目标 期间是在DNA测序中实施四衰变检测。 同时，研究将针对寿命的优化 检测和提高检测限，这将通过（1） 可见光染料的进一步研究和能量传递的新研究 策略和近红外染料，（2）设计和实现了一个 共焦光学检测系统;（3）CE/MHF接口的优化 设计，以及（4）不同CE包装材料和缓冲液的研究 以增强毛细管上荧光寿命检测。

DESCRIPTION (Adapted from the Investigator's Abstract): A new scheme for detection of electrophoretically separated, fluorescent-labeled DNA fragments for genome sequencing is under investigation. In this multiplex scheme, fluorescence lifetime replaces spectra selectivity for distinguishing between the four base-specific labels. Thus, the scheme is based on detection of four fluorescence decays instead of four colors in a single-lane separation of the DNA fragments. Fluorescence lifetime is a concentration-independent characteristic that provides "digital" detection, offering important advantages over analog, intensity-dependent measurements. Since the fluorescence lifetimes of the four dyes are pre-determined, data analysis is simple, straightforward even, when there is incomplete electrophoretic resolution. Continuous, on-the-fly fluorescence lifetime detection in four-decay sequencing is performed in the frequency-domain using a state-of-the-art, commercial instrument that incorporates multiharmonic Fourier transform (MHF) technology to allow measurements of phase and modulation simultaneously at many modulation frequencies. The MHF instrument is interfaced to a commercial capillary electrophoresis (CE) system. Fluorescence lifetime and intensity are simultaneously derived from the same measurement to produce a lifetime-intensity electropherogram in no more time than is required for a simple intensity or color electropherogram. In fact, the improved accuracy and resolution that is expected from lifetime detection should enable faster sequencing since the demands on the electrophoretic resolution will be eased. The goal for the next project period is the implementation of four-decay detection in DNA sequencing. Concurrently, studies will be directed towards optimization of lifetime detection and improved detection limits, which will be achieved through (1) further investigations of visible dyes and new studies of energy-transfer strategies and near-infrared dyes, (2) design and implementation of a confocal optical detection system, (3) optimization of the CE/MHF interface design, and (4) investigations of different CE packing materials and buffers to enhance on-capillary fluorescence lifetime detection.