ENHANCED INSTRUMENTATION FOR DETECTING FLUORESCENT DNA

以及时和成本有效的方式对人类基因组进行测序将 需要改进的仪器。该项目的目标是提高 现有Marshfield扫描荧光检测器的性能 （SCAFUD）用于DNA测序的目的。SCAFUD用于检测 当荧光标记的DNA片段迁移通过144泳道时， 垂直聚丙烯酰胺变性平板凝胶。荧光信号来自 探测器被排列成计算机图像文件， 使用一套图像分析程序进行操作。现有SCAFUD 具有63 × 10-6核苷酸测序的最大理论输出 每一年。本提案中描述的改进将提高最大值 理论产量至少为215 × 10-6核苷酸/年。 测序性能提高约3.5倍， 通过一些增量硬件和软件实现 改进.仪器设计将合并和模块化， 降低故障率，简化维修。一个新的质量更小， 将开发仪器的不透光盖。自动对焦将 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， 连接到每个探测器。将引入激发激光 通过布鲁斯特角进入凝胶，与当前的epi相比， 照明设计输入激光的偏振和滤波 将被测试作为一种手段，减少背景光到达 探测器从每个SCAFUD获得的序列信息量 运行将通过在前进和前进期间收集数据来增加， 激光头的返回通道。凝胶厚度、长度、组成和 将改变温度以增加质量序列 可以收集信息。可检测的染料数量 同时将通过添加第二个，可能的话， 第三激光源，并通过显著简化检测系统。 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 变化测序成本的最小化将是压倒一切的因素 用于评价所有仪器修改。 现有和未来的图像分析和碱基调用软件将 一般化并记录，以便于将该软件转移到 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， 一个类似的文书，由一个私人公司和/或通过释放 仪表的详细结构图和零件清单， 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.