ENHANCED INSTRUMENTATION FOR DETECTING FLUORESCENT DNA

以及时和具有成本效益的方式对人类基因组进行测序将 需要改进仪器设备。该项目的目标是增强 现有马什菲尔德扫描荧光探测器的性能 (SCAFUDS)用于DNA测序。SCAFUD用于检测 荧光标记的DNA片段在144巷迁移时， 垂直、聚丙烯酰胺变性平板凝胶。来自中国的荧光信号 探测器被排列成计算机图像文件，然后 使用一套图像分析程序进行操作。现有SCAFUD 测序的最大理论输出为63×10-6个核苷酸 每年。本提案中所述的改进将提高最大 理论产量至少达到每年215×10-6个核苷酸。 测序性能大约提高3.5倍将是 通过多个增量硬件和软件实现 改进。仪器设计将统一和模块化，以 降低故障率，简化维修。一个不那么大而更大的新 将开发该仪器的轻便密封罩。自动对焦将 实施以减少仪器变异性、操作员错误，并 提高激光安全性。将设计可调增益放大器，并 连接到每个探测器上。本课程将介绍激发激光 通过布鲁斯特角进入凝胶，与目前的表观电位相比- 照明设计。输入激光的偏振和滤光 将作为一种减少到达 探测器。从每个SCAFUD获得的序列信息量 通过在转发和转发期间收集数据，将增加运行 激光头的返回通道。凝胶厚度、长度、成分和 温度将改变，以提高质量序列的速度 信息是可以收集的。可检测到的染料数量 同时将通过增加第二个，并可能， 第三种激光光源，大大简化了检测系统。 将广泛使用新的多通道探测器阵列。图像 分析软件将根据需要进行修改以调整硬件的速度 改变。将测序成本降至最低将是压倒一切的因素 用于所有仪器改装的评估。 现有和未来的图像分析和基础调用软件将 通用化和文档化，以便将本软件传输到 其他研究实验室。将SCAFUD硬件技术转让给 其他研究实验室将通过市场营销来完成 由一家私人公司和/或通过发放 仪器的详细施工图和部件列表，请参阅 马什菲尔德。

Sequencing of the human genome in a timely and cost effective manner will require improved instrumentation. The goal of this project is to enhance the performance of existing Marshfield SCAnning FlUorescence Detectors (SCAFUDs) for the purpose of DNA sequencing. SCAFUDs are used to detect fluorescently labeled DNA fragments as they migrate through 144 lane, vertical, polyacrylamide denaturing slab gels. Fluorescent signals from the detectors are arrayed into computer image files which are then manipulated using a suite of image analysis programs. Existing SCAFUDs have a maximum theoretical output for sequencing of 63 x 10-6 nucleotides per year. Improvements described in this proposal will boost the maximum theoretical output to at least 215 x 10-6 nucleotides per year. The approximately 3.5 fold increase in sequencing performance will be achieved through a number of incremental hardware and software improvements. Instrument design will be consolidated and modularized to reduce failure rate and to simplify repair. A new less massive and more light tight cover for the instrument will be developed. Autofocusing will be implemented to reduce instrument variability, operator error, and to improve laser safety. Adjustable gain amplifiers will be designed and attached to each detector. The excitation laser light will be introduced into the gel through Brewster's angle as compared to the current epi- illumination design. Polarization and filtering of the input laser light will be tested as a means of reducing background light reaching the detectors. The amount of sequence information obtained from each SCAFUD run will be increased by collecting data during both the forward and return passes of the laser head. Gel thickness, length, composition, and temperature will be varied to increase the rate at which quality sequence information can be collected. The numbers of dyes which can be detected simultaneously will be increased by addition of a second and, possibly, third laser source and by significantly simplifying the detection system. Extensive use will be made of new multichannel detector arrays. Image analysis software will be modified as necessary to pace the hardware changes. Minimization of sequencing cost will be the overriding factor used in evaluation of all instrument modifications. Existing and future image analysis and base calling software will be generalized and documented to facilitate transfer of this software to other research laboratories. Transfer of the SCAFUD hardware technology to other research laboratories will be accomplished either through marketing of a similar instrument by a private firm and/or through release of detailed construction drawings and parts lists for the instrument from Marshfield.