A kit to detect and count individual DNA molecules of interest

描述（由申请人提供）：核酸浓度的准确测量在基因组研究的许多领域及其对人类健康的应用中是必不可少的。在许多重要的公共卫生问题中，包括癌症、传染病和产前诊断，灵敏、精确和可靠的定量核酸检测越来越有必要。然而，目前缺乏能够进行高精度测量的实用技术，这在必须辨别非常小的浓度差异的研究和临床应用中产生了未满足的需求。 我们最近开发了一种具有数字PCR统计能力的高灵敏度计数方法，但可以同时多重测量许多基因。在这个提议中，我们将把这项技术发展成一个单分子检测系统，它将允许对核酸的单拷贝进行绝对计数。对于该提案的第一阶段，我们的目标是开发一种原型检测试剂盒和检测器，以精确测量基因表达。 虽然现代分子生物学中的许多技术允许核酸的相对定量，但数字PCR是唯一能够绝对定量的方法。然而，它需要昂贵的试剂和仪器，并且仅限于一次测量单个目标。相比之下，我们的新方法首先用寡核苷酸条形码标签随机标记DNA分子的每个拷贝。PCR扩增后，构建互补寡核苷酸的小检测器面板以检测不同条形码标签的数量，其揭示溶液中原始分子的数量。这将计数相同DNA的单个分子的困难任务转化为检测存在的不同扩增条形码序列标签的数量的简单过程。由于我们的方法将相同的分子扩展到化学空间，因此我们可以在单个试管中进行测量，这使得它比需要大量单独容器的数字PCR更简单且易于进行多重目标检测。将检测已知浓度的对照核酸，并使用数字PCR进行独立测量，以验证该技术。 我们公司由一支非常强大的成功创新者和科学家/工程师团队组成，在研究和产品商业化方面都取得了重大成就。此外，我们还与斯坦福大学基因组技术中心的科学家建立了合作关系，使我们能够使用这个世界一流研究机构提供的仪器和专业知识。

DESCRIPTION (provided by applicant): Accurate measurement of nucleic acid concentrations is essential in many areas of genome research and its application to human health. Sensitive, precise and reliable quantitative nucleic acid tests are increasingly necessary in many important public health problems, including cancer, infectious disease and prenatal diagnostics. However, there is currently a lack of practical technologies capable of high precision measurements, creating an unmet need in research and clinical applications where very small differences in concentration must be discerned. We have recently developed a high-sensitivity counting method with the statistical power of digital PCR, yet can be multiplexed to measure many genes simultaneously. In this proposal, we will develop this technology into a single molecule detection system which will allow absolute counting of single copies of nucleic acids. For phase I of this proposal, our objective is to develop a prototype assay kit and detector to make precise measurements of gene expression. Although many techniques in modern molecular biology allow for the relative quantitation of nucleic acids, digital PCR is the only method capable of absolute quantitation. However, it requires expensive reagents and instruments, and is limited to measurements of a single target at a time. In contrast, our novel method first randomly labels every copy of a DNA molecule with an oligonucleotide barcode tag. After PCR amplification, a small detector panel of complementary oligonucleotides is constructed to detect the number of different barcode tags, which reveals the number of original molecules in solution. This transforms the difficult task of counting individual molecules of identical DNAs into the simple process of detecting the number of different amplified barcoding sequence tags present. Because our method expands identical molecules into chemical space, we can perform the measurement in a single tube, making it much simpler and amenable to multiplex target detection than digital PCR where a very large number of separate containers is required. Control nucleic acids of known concentrations will be tested, and independent measurements will be conducted with digital PCR in order to validate the technique. Our company is comprised of an exceptionally strong team of successful innovators and scientists/engineers with significant achievements in both research and product commercialization settings. Additionally, we have established collaborations with scientists at the Stanford University Genome Technology Center, giving us access to instruments and expertise available at this world class research facility.