Massively Parallel Cloning and Sequencing of DNA

描述(申请人提供)：我们的研究目标是开发两项创新技术：大规模并行全基因组扩增和DNA变性测序(SBD)。拟议的研究将解决对开发下一代测序技术至关重要的两个技术问题：1)开发从整个基因组中平行克隆扩增单个DNA分子的方法；2)开发一种超高通量测序策略，该策略可以在一个集成的微型设备中将基因组规模的样本扩增和处理集成到测序工作流程中。我们已经证明，使用一种名为滚环扩增(RCA)的强大的等温DNA扩增技术，可以在固体载体上大规模平行分离和克隆数亿个单环DNA分子。我们还制定了一个概念性的框架，用于“排序依据” 变性“技术，实现快速、准确的DNA测序。我们提出了用滚环扩增技术在单芯片上从哺乳动物大小的整个基因组中分离和克隆单个弹枪DNA片段的可行性。我们还将演示用于高通量DNA测序的新的“变性测序”方法的原理证明。完成拟议的里程碑将为一个集成系统奠定技术框架，该系统将使全基因组扩增和测序能够在单一的微型设备中进行。通过从根本上消除昂贵的试剂和传统的克隆、克隆和其他过程的需要，基因组测序成本可以减少几个数量级。从长远来看，我们的技术将具有巨大的潜力来实现这种RFA的目标，即非常便宜的基因组重新测序。一旦这种集成的台式系统完全开发出来，基因组测序可能会成为许多单独研究和诊断实验室的常规操作。个人基因组计划可能成为现实。这将真正改变生物医学研究和个人医疗保健的性质。

DESCRIPTION (provided by applicant): The objective of our research is to develop two innovative technologies: massively parallel whole genome amplification and DNA sequencing by denaturation (SBD). The proposed research will address two technological issues critical for the development of the next-generation sequencing technologies: 1) the development of methods for the parallel clonal amplification of individual DNA molecules from whole genomes; and 2) the development of an ultra high throughput sequencing strategy that can integrate genome-scale sample amplification and processing into the sequencing workflow in an integrated miniaturized device. We have demonstrated that hundreds of millions of single circular DNA molecules can be separated and cloned in massive parallel on solid supports using a powerful isothermal DNA amplification technique called rolling circle amplification (RCA). We have also developed a conceptual framework for the "sequencing by denaturation" technology for rapid and accurate DNA sequencing. We propose to demonstrate the feasibility of separating and cloning individual shot-gun DNA fragments from a whole mammalian-size genome in a small area on a single chip using the rolling circle amplification technology. We also will demonstrate the proof-of-principle of the novel "sequencing by denaturation" method for high throughput DNA sequencing. Accomplishing the proposed milestones will lay down a technological framework for an integrated system that will enable whole genome amplification and sequencing to be carried out in a single miniaturized device. Genome sequencing cost can be reduced by several orders of magnitude by essentially eliminating the needs for costly reagents and conventional cloning, colony picking and other processes. In the long run our technology will have a great potential to achieve the goal of this RFA for very inexpensive re-sequencing of genomes. Once such an integrated bench-top system is fully developed, genome sequencing could be routine operations of many individual research and diagnostic laboratories. The personal genome project could be a reality. That will truly transform the nature of biomedical research and individual healthcare.