DEVELOPMENT OF A JBS HI-LO URINE DNA KIT

 描述(由申请人提供)：此SBIR第一阶段申请是对题为“从易于收集和存储的人类样本中获取全面基因组信息的开发方法”的计划公告PAR-13-203的响应。这项建议特别针对从尿液中获取高质量和全面的基因组数据的敏感和成本效益技术的要求，尿液是一种易于收集和易于处理的人体样本形式。我们已经制定了为基因研究收集尿液的标准操作程序(SOP)，可用于研究目的，已在NCI EDRN资助的验证研究(方案ID320)中实施。我们还开发了一种获得专利的分离方法，使我们能够根据片段大小将DNA分成两个不同的物种。大于1kb的DNA称为高分子量(HMW)DNA，主要来源于尿路细胞碎片。相反，从100到400个碱基的DNA被指定为低分子量(LMW)DNA，它主要来自循环。我们已经证明，如果肿瘤存在，LMW DNA可以用于检测肿瘤循环衍生的遗传标记。在第一阶段，本申请的目标1是证明HMW尿液DNA可以取代全血作为DNA来源，以获得全面和高质量的基因组信息。我们还将证明，低分子量尿DNA可以作为无细胞凋亡DNA的来源，用于检测整个身体的体细胞/获得性遗传变异，类似于血浆DNA(目标2)。我们建议使用TP53密码子249突变作为体细胞突变标记，以比较LMW尿DNA和血浆DNA对检测循环衍生突变的敏感性。在第二阶段，我们建议开发尿液DNA收集试剂盒(JBS Hi-Lo尿液DNA试剂盒)，以优化尿液的使用，该试剂盒既易于收集，又易于携带(包括样本运送和储存)，供临床使用。预计这项建议将导致开发一种试剂盒，该试剂盒将提供完整的端到端解决方案，涵盖从样本采集、核酸提取到测序的所有步骤，以获得完整的基因组信息，在临床和研究中具有各种应用。

 DESCRIPTION (provided by applicant): This SBIR phase I application is being submitted in response to the Program Announcement PAR-13-203 entitled "Methods of Development for Obtaining Comprehensive Genomic Information from Human Specimens that are Easy to collect and store." This proposal specifically addresses the request for sensitive and cost-effective technologies for obtaining high-quality and comprehensive genomic data from urine, which is both an easy to collect and easy to handle form of human specimen. We have already developed the standard operating procedures (SOP) to collect urine for genetic studies, which can be used for research purposes that have been implemented in the NCI EDRN-funded validation study (protocol ID320). We have also developed a patented fractionation method that enables us to separate the DNA into two distinct species based on fragment sizes. DNA larger than 1 kb is designated as high molecular weight (HMW) DNA, which is mostly derived from the cell debris of the urinary tract. Conversely, the DNA ranging from 100 to 400 bp is designated as low molecular weight (LMW) DNA, which is derived mostly from the circulation. We have shown that LMW DNA can be used for the detection of circulation-derived genetic markers of cancer, if the tumor is present. In phase I, Aim 1 of this application is to demonstrate that HMW urine DNA can replace whole blood as a DNA source for obtaining comprehensive and high-quality genomic information. We will also demonstrate that LMW urine DNA can be a source for cell-free apoptotic DNA for detecting somatic/acquired genetic variants from the entire body, similar to plasma DNA (Aim 2). We propose to use TP53 codon 249 mutations as a somatic mutation marker to compare the LMW urine DNA and plasma DNA for the sensitivity of detecting circulation-derived mutations. In phase II, we propose to develop the urine DNA collection kit (JBS Hi-Lo urine DNA kit) to optimize the use of urine, which is both easy to collect and easy to handle (including sample transportation and storage) for clinical use. It is anticipated that this proposal will lead to the development of a kit that will provide a complete end-to-end solution that addresses all the steps from sample collection and nucleic acid extraction to sequencing, for obtaining complete genomic information with various applications in both clinic and research.