Massively Parallel Cloning and Sequencing of DNA

描述（由申请人提供）：我们的研究目标是开发两种创新技术：大规模并行全基因组扩增和变性DNA测序（SBD）。拟议的研究将解决两个关键的技术问题，为下一代测序技术的发展：1）从整个基因组的单个DNA分子的平行克隆扩增的方法的发展;和2）超高通量测序策略的发展，可以集成基因组规模的样品扩增和处理到测序工作流程中的集成微型设备。 我们已经证明，使用称为滚环扩增（RCA）的强大的等温DNA扩增技术，可以在固体支持物上大规模平行地分离和克隆数亿个单个环状DNA分子。我们还制定了一个概念框架， 利用“变性”技术进行快速准确的DNA测序。我们建议使用滚环扩增技术在单个芯片上的小区域中从整个人大小的基因组中分离和克隆单个鸟枪DNA片段的可行性。我们还将证明新的“变性测序”方法的原理，用于高通量DNA测序。完成拟议的里程碑将为一个综合系统奠定技术框架，使整个基因组扩增和测序能够在一个小型化装置中进行。通过基本上消除对昂贵试剂和常规克隆、菌落挑选和其他过程的需要，基因组测序成本可以降低几个数量级。从长远来看，我们的技术将有很大的潜力实现RFA的目标，对基因组进行非常便宜的重新测序。一旦这样一个集成的台式系统被完全开发出来，基因组测序可能是许多个体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.