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：大规模平行全基因组扩增和DNA变性测序（SBD）。本研究将解决对下一代测序技术发展至关重要的两个技术问题：1)开发从全基因组中克隆扩增单个DNA分子的方法；2)开发一种超高通量测序策略，将基因组尺度的样品扩增和处理集成到一个集成的小型化设备的测序工作流程中。我们已经证明，使用一种强大的等温DNA扩增技术，称为滚动圈扩增（RCA），可以在固体支架上大量平行地分离和克隆数亿个单环DNA分子。我们还开发了一个“按顺序排序”的概念框架

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.