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

描述(申请人提供)：串联重复序列(STR)广泛分布于人类基因组中。这些位点表现出高度的长度多态，这是因为它们在DNA复制过程中相对不稳定，即在模板和新合成的链之间发生滑动的倾向，导致重复单位的缺失或增加。STR重复数的变化可能导致可遗传的遗传易感性和疾病。然而，它们的可变性使它们成为遗传学研究的良好标记物。一类是包含4个碱基对重复单位的微卫星，已被联邦调查局选为名为CODIS的遗传识别系统的基础。目前的分析系统需要1-2天的分析，并使用包含STR基因座的DNA片段的电泳法分离，以准确确定重复数。 该项目的长期目标是开发一种用于STR分析的简单、成本效益高和自动化的分析系统，通过避免需要电泳分离和潜在地避免需要PCR扩增，使得从样本到结果的人类识别不到2小时。在这项建议的第一阶段，我们将使用单基因座(TPOX)来证明基于微阵列的准确的STR分析在技术上是可行的，并且足够快速。在第二阶段，我们将扩大这项检测以包括其他几个CODIS基因座，并试图通过取消PCR步骤来进一步提高检测速度。 我们实现这些目标的方法结合了两家公司的技术。Gene Check公司开发了RML技术，其中RecA催化STR基因座上的寡核苷酸探针的特定碱基配对，然后连接到反映STR基因座长度的特定产物中。每一种产物都有一个独特的识别序列编码，这使得它们可以通过杂交到微阵列来分离。纳米球已经开发了一个基于微阵列的自动化分析平台，具有足够的特异性和灵敏度，可以通过使用纳米颗粒检测技术从总基因组DNA中检测SNP，而不需要进行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。这将为拟议的将这项技术扩展到其他几个FBI CODIS STR基因座的第二阶段奠定基础，并为避免PCR的分析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项技术在医疗应用中的潜力，例如组织分型、移植监测和由相对短的核苷酸重复多态介导的遗传条件或易感性的分析。

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.