GENOME SEQUENCING BY 'FOUR-DECAY' FLUORESCENCE DETECTION

提出了一种新的方案， 用于基因组测序的荧光标记的DNA片段，其中 荧光寿命取代光谱选择性， 在四个碱基特异性标签之间。因此，该方案基于 四种荧光衰减的检测，而不是在一个单一的四种颜色， DNA片段的泳道分离。与模拟性质相反， 强度（“颜色”）检测，荧光寿命是浓度- 提供数字检测的独立特性， 色谱检测的重要优势。而且由于 四种染料的荧光寿命将是已知的，数据分析将 简单明了，即使不完整， 色谱分离度 将进行连续、实时荧光寿命检测 在频域中使用最先进的商业仪器 它结合了多谐波傅里叶变换技术， 在多个调制下同时测量相位和调制 频率.荧光强度同时来源于 同样的测量产生寿命-强度色谱图， 时间比传统的强度色谱所需的时间， 用于四色检测方案。事实上，改进的分辨率 寿命检测的预期结果应该能够实现更快的测序 因为对色谱分辨率的要求将被减轻。 拟议的研究将分三个阶段进行。第一，候选人 必须确定四种碱特异性染料。染料应该有 相似的光谱特性和可分辨的寿命，以及 符合与可探测性、灵敏度、化学 反应性和电泳迁移率。除了可见的染料， 主要是氧杂蒽类，近红外（NIR）染料也将是 研究了在近红外工作的优点是低 背景信号和非常低成本的仪器。一旦一些有前途的 染料已被选定，他们将单独研究和混合物 电泳凝胶，以表征其光谱和 寿命特性。检测极限和寿命分辨率将是 为了进一步缩小可能性，最后 生产几个四染料组用于进一步研究。最后阶段 将实现对标记DNA的实时寿命检测， 从标记的核苷酸开始， 片段，并最终进展到更长的 寡核苷酸

A new scheme is proposed for detection of electrophoretically separated, fluorescent-labelled DNA fragments for genome sequencing, in which fluorescence lifetime replaces spectral 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. In contrast to the analog nature of intensity ("color") detection, fluorescence lifetime is a concentration- independent characteristic that provides digital detection, offering important advantages for chromatographic detection. Moreover, since the fluorescence lifetimes of the four dyes will be known, data analysis will be simple and straightforward even when there is incomplete chromatographic resolution. Continuous, on-the-fly fluorescence lifetime detection will be performed in the frequency-domain using a state-of-the-art, commercial instrument that incorporates multiharmonic Fourier transform technology to allow measurements of phase and modulation simultaneously at many modulation frequencies. Fluorescence intensity is simultaneously derived from the same measurement to produce a lifetime-intensity chromatogram in no more time than is required for a conventional intensity chromatogram such as is used in the four-color detection scheme. In fact, the improved resolution that is expected from lifetime detection should enable faster sequencing since the demands on the chromatographic resolution will be eased. The proposed research will be performed in three phases. First, candidates for the four base-specific dyes must be identified. The dyes should have similar spectral characteristics and resolvable lifetimes as well as meeting other criteria related to detectability, sensitivity, chemical reactivity and electrophoretic mobility. In addition to visible dyes, primarily of the xanthene group, near-infrared (NIR) dyes will also be investigated. Among the advantages of working in the NIR are low background signal and very low cost instrumentation. Once some promising dyes have been selected, they will be studied individually and in mixtures on electrophoresis gels in order to characterize their spectral and lifetime properties. Limits of detection and lifetime resolution will be determined in order to further narrow down the possibilities, finally producing a few four-dye sets for further investigation. The final phase will be implementation of on-the-fly lifetime detection of labelled DNA, beginning with labelled nucleotides and progressing to short labelled fragments, and eventually progressing to actual sequencing of longer oligonucleotides.