GENOME SEQUENCING BY FOUR DECAY FLUORESCENCE DETECTION

描述(改编自调查者摘要)：一种新的方案 电泳法分离的荧光标记DNA的检测 用于基因组测序的片段正在调查中。在这个影院里 方案，荧光寿命取代光谱选择性 区分四个碱基特定的标记。因此，该方案是 基于对四种荧光衰减而不是一种颜色的检测 DNA片段的单通道分离。荧光寿命是一个 不依赖于浓度的特性，提供“数字”检测， 与依赖强度的模拟测量相比，具有重要的优势。 由于这四种染料的荧光寿命是预先确定的，数据 分析是简单的，甚至是直截了当的，当存在不完整的时候 电泳法分辨率。连续的动态荧光寿命 四衰变序列中的检测是在频域中执行的 使用最先进的商业工具，结合了 多次谐波傅里叶变换(MHF)技术允许测量 在许多调制频率下同时进行相位和调制。MHF 仪器与商用毛细管电泳仪(CE)接口 系统。荧光寿命和强度同时从 用同样的方法在NO中产生寿命强度的电泳图 比简单的强度或彩色电泳图所需的时间更长。 事实上，更高的精确度和分辨率是生命周期所期望的 检测应该能够实现更快的排序，因为对 电泳法的分辨率将会降低。下一个项目的目标 周期是DNA测序中四衰变检测的实现。 同时，研究的方向将是优化寿命 检测和改进检测限值，这将通过(1)实现 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 共焦光学探测系统；(3)CE/MHF接口的优化 设计，以及(4)不同CE包装材料和缓冲器的研究 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.