权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

SOLID STATE COSMID FINGERPRINTING

固态粘胶指纹图谱

基本信息

批准号：
2459847
负责人：
JAN H HOH
金额：
$ 15.44万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1996
资助国家：
美国
起止时间：
1996-08-01 至 1999-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2459847
关键词：
atomic force microscopy computer program /software genetic techniques method development plasmids restriction fragment length polymorphism

项目摘要

DESCRIPTION (Adapted from the Investigator's Abstract): The nucleotide sequence of the human genome will provide health benefits ranging from new diagnostic tools to therapeutic reagents. The broad long term goal of the present proposal is to address one technical aspect of the human genome project, that of generating ordered sets of cosmids or similar sized DNA segments for sequencing. To achieve this goal it is proposed to develop an automated high throughput solid state fingerprinting (SSFP) method based on atomic force microscopy (AFM). The proposed method has several important features. (1) It is a highly automated solid state method; (2) the order of restriction fragments is maintained; (3) the estimated throughput is 170,000 cosmids per year per AFM; (4) sub-nanogram amounts of DNA are required for a fingerprint; and (5) the cost is relatively low (less than $0.5 per cosmid). The AFM has a well-demonstrated ability to determine the lengths of DNA fragments ranging from about 50 to 50,000 base pairs, and to image proteins bound to DNA. Taking advantage of this capability, several hundred cosmids will be attached to a surface and the position of restriction sites in each one will be marked by digesting the DNA with a restriction enzyme, causing a small break in the strand, or decorating the DNA with inactive restriction enzymes. The size and order of the restriction fragments will be determined automatically by AFM combined with special pattern recognition software, to provide a characteristic fingerprint that can be used to determine overlap of the cosmids. The present proposal has three specific aims directed toward the development of this system: (1) development of sample preparation and labeling methods that are suitable for large scale automation. The investigators will use the vinyl-based covalent attachment of DNA to a silicon surface, and subsequent alignment of the DNA on the surface, to prepare several hundred samples in parallel. (2) Optimization of the data acquisition conditions. The maximal scan speeds, image size, and the best imaging environment for high throughput imaging will be determined. (3) Development of automated data analysis methods of generating DNA fingerprints. The investigators will use pattern recognition methods to determine the size and position of restriction fragments. The software will be made as robust as possible, in order to accommodate background and image quality variations.

描述（改编自研究者摘要）：核苷酸人类基因组序列将提供健康益处，诊断工具到治疗试剂。的广泛的长期目标目前的建议是解决人类基因组的一个技术问题项目，即生成有序的粘粒或类似大小的DNA集的项目用于测序的片段。为了实现这一目标，建议制定一项自动化高通量固态指纹（SSFP）方法，基于原子力显微镜（AFM）。所提出的方法有几个重要的功能. (1)这是一种高度自动化的固态方法;（2）顺序保留限制性片段;（3）估计吞吐量为170，000 （4）每一个原子力显微镜每年需要亚纳克量的DNA，指纹;和（5）成本相对较低（每个粘粒低于0.5美元）。原子力显微镜有一个很好的证明能力，以确定DNA的长度片段范围从大约50到50，000个碱基对，与DNA结合。利用这种能力，数百名宇航员将附着在表面上，并且限制性位点在每一个中的位置一种是用限制性内切酶消化DNA，链中的小断裂，或用非活性限制修饰DNA 内切酶将确定限制性片段的大小和顺序自动由原子力显微镜结合专用的模式识别软件，提供可用于确定重叠的特征指纹宇宙的。本建议有三个具体目标，对本系统的开发：（1）样本的开发适合大规模生产的制备和标记方法自动化. 研究人员将使用乙烯基共价连接 DNA的硅表面，和随后的DNA上的对齐，表面，平行制备数百个样品。 (2)优化数据采集条件。最大扫描速度、图像尺寸、高通量成像的最佳成像环境将是测定 (3)自动化数据分析方法的开发生成DNA指纹研究人员将使用模式识别确定限制性片段大小和位置的方法。的软件将尽可能强大，以适应背景和图像质量变化。