A microarray based STR genotyping system utilizing RecA-mediated ligation and nan

描述（由申请人提供）：串联重复序列（STR）广泛分布于人类基因组中。这些位点表现出高水平的长度多态性，这是它们在DNA复制过程中相对不稳定的结果，即，在模板和新合成的链之间发生滑动的趋势，导致重复单元的缺失或添加。STR重复序列数的变化可导致遗传性遗传倾向和疾病。然而，它们的变异性使它们成为遗传研究的良好标记。一类是含有4个碱基对重复单位的微卫星，已被联邦调查局选为遗传识别系统（称为CODIS）的基础。目前的分析系统需要1-2天的测定，并采用含有STR基因座的DNA片段的电泳分离，以准确确定重复数的大小和鉴定。 该项目的长期目标是开发一种简单，具有成本效益和可自动化的STR分析系统，通过避免电泳分离的需要和可能避免PCR扩增的需要，允许在不到2小时的时间内从样本到结果进行人类识别。在本建议的第一阶段，我们将使用一个单一的基因座（TPOX），以证明准确的微阵列为基础的STR分析在技术上是可行的，足够快。在第二阶段，我们将扩大这一检测，包括其他几个CODIS基因座，并试图通过消除PCR步骤，以提高检测速度进一步。 我们实现这些目标的方法结合了两家公司的技术。基因检查公司开发了RML技术，其中RecA催化STR基因座处寡核苷酸探针的特异性碱基配对，随后连接成反映STR基因座长度的特异性产物。每种产物都用独特的识别序列编码，这使得它们可以通过与微阵列杂交来分离。Nanosphere开发了一种基于自动化微阵列的检测平台，具有足够的特异性和灵敏度，可以通过采用纳米颗粒检测技术从总基因组DNA中检测SNP，而无需PCR扩增。 我们的初步数据表明，第一阶段的任务可以在6-12个月内完成。这将为该技术拟议的第二阶段扩展到几个额外的FBI CODIS STR基因座以及避免PCR的检测和系统设计奠定基础。此外，这些结果应该使我们能够评估这种技术在医学应用中的潜力，例如组织分型，移植监测和分析由相对较短的核苷酸重复多态性介导的遗传条件或易感性。

DESCRIPTION (provided by applicant): Tandem repeat sequences (STR) are distributed widely in the human genome. These sites exhibit high levels of length polymorphism, as a result of their relative instability during DNA replication, i.e., the tendency for slippage to occur between the template and newly synthesized strands causing deletion or addition of repeat units. Changes in STR repeat number can lead to heritable genetic predispositions and disease. However, their variability makes them good markers for genetic studies. One class, the microsatellites containing 4 base pair repeat units, has been selected by the FBI as the basis for a genetic identification system, termed CODIS. Current analysis systems require a 1-2 day assay and employ electrophoretic separation of DNA fragments that contain the STR locus for accurate sizing and identification of the repeat number. The long-term goal of this project is to develop a simple, cost-effective and automatable assay system for STR analysis that allows human identification in less than 2 hours from sample to result by avoiding the need for electrophoretic separation and potentially avoiding the need for PCR amplification. In Phase I of this proposal we will use a single locus (TPOX) to demonstrate that accurate microarray based STR analysis is technically feasible and sufficiently rapid. In Phase II we will expand this assay to include several other CODIS loci, and attempt to increase assay speed further by eliminating the PCR step. Our approach to achieving these goals combines the technologies of two companies. Gene Check, Inc. has developed RML technology in which RecA catalyzes specific base pairing of oligonucleotide probes at the STR locus with subsequent ligation into specific products that reflect the length of the STR locus. Each product is coded with a unique recognition sequence, which allows their separation by hybridization to a microarray. Nanosphere has developed an automated microarray-based assay platform with sufficient specificity and sensitivity to allow SNP detection from total genomic DNA without PCR amplification by employing nanoparticle detection technology. Our preliminary data suggest that the Phase I tasks can be accomplished within a 6-12 month period. This will lay the groundwork for the proposed Phase II extension of this technology to several additional FBI CODIS STR loci, and assay and system designs to avoid PCR. Moreover, the results should allow us to assess the potential of this technology for medical applications, such as tissue typing, transplantation monitoring, and analysis of genetic conditions or predispositions mediated by relatively short nucleotide repeat polymorphisms.