Microarray based STR genotyping utilizing RecA-mediated ligation

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。这些位点由于它们在DNA复制过程中的相对不稳定性而表现出高水平的长度多态性，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理想标记。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、经济高效且可自动化的STR分析检测系统，通过避免PCR扩增、靶DNA变性和电泳分离，使人类识别从样本到结果在3小时内完成。在该提案的第一阶段，使用单个基因座（TPOX）来证明基于精确微阵列的STR分析在技术上是可行的并且足够快速。在第二阶段，我们将扩大这一检测方法，包括至少6个其他CODIS基因座，将技术从溶液中转化为固定化形式，并试图通过消除PCR步骤进一步提高检测速度和便利性。我们实现这些目标的方法利用Gene Check的RecA介导的连接（RML）技术，其中RecA介导的双链DNA同源性搜索的极端精确性用于催化STR基因座处的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。Gene Check已经发现，当其中一种寡核苷酸固定在微阵列载玻片上时，RML可以有效地操作，使得RML-STR技术的高阶多重化可行且实用。将采用信号放大方法，以允许直接从双链基因组DNA进行基因分型。该项目的成功将产生一个STR小组，可以立即用于法医应用以及人类亲子鉴定和身份测试。RML-STR系统可以容易地适用于家畜、伴侣动物和野生动物的亲子鉴定和身份检验，并且具有潜在的应用于与微卫星序列扩增相关的医学病症的诊断。 公共卫生相关性： Gene Check提出利用其RecA介导的连接技术以微阵列形式进行STR基因分型（RML-STR），以实现稳健、快速、低成本和自动化的STR基因分型。该系统将有助于日常警察调查中的人员身份识别以及各种其他法医和非人类身份识别应用。医学应用包括三联体重复序列相关疾病的组织分型和遗传分析。

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 ideal 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 3 hours from sample to result by avoiding the need for PCR amplification, target DNA denaturation and electrophoretic separation. In Phase I of this proposal a single locus (TPOX) was used to demonstrate that accurate microarray based STR analysis is technically feasible and sufficiently rapid. In Phase II we will expand this assay to include at least 6 other CODIS loci, convert the technology from an in solution to an immobilized format and attempt to increase assay speed and convenience further by eliminating the PCR step. Our approach to achieving these goals utilizes Gene Check's RecA mediated ligation (RML) technology in which the extreme precision of RecA mediated homology searching in double stranded DNA is used to catalyze specific base pairing of oligonucleotide probes at the STR locus with subsequent ligation into specific products that reflect the length of the STR locus. Gene Check has discovered that RML can operate efficiently when one of the oligonucleotides is immobilized to a microarray slide, making high order multiplexing of the RML-STR technology feasible and practical. Signal amplification methods will be employed to allow genotyping directly from double stranded genomic DNA. Success in this project will produce an STR panel that can immediately be used in forensic applications as well as in human paternity and identity testing. The RML-STR system can readily be adapted to livestock, companion animal and wild animal parentage and identity testing and has potential application to diagnosis of medical conditions related to microsattelite sequence expansion. PUBLIC HEALTH RELEVANCE: Gene Check proposes to utilize its RecA mediated ligation technology for STR genotyping (RML-STR) in a microarray format to allow robust, rapid, low cost and automated STR genotyping. This system will be useful for personnel identification in everyday police investigations as well as in a variety of other forensic and non-human identification applications. Medical applications include tissue typing and genetic analysis of triplet repeat associated disorders.