Isolation Selection and Polony Amplification of Single Cells in a Gel Matrix

描述(由申请人提供)：人类微生物组代表了一个在人类健康和疾病中可能发挥作用的基本上未定义的生物体联合体。为了更好地了解人类宿主的复杂微生物生态，需要新的技术来分离和排列个体的“参考”基因组。具体目的：1)测试在聚丙烯酰胺水凝胶中使用固相PCR从单细胞全基因组中分离和扩增多聚酶菌落(Polonies)的机制；以及2)探索紫外光催化作为一种选择性地削弱微生物细胞壁的方法，从而使DNA聚合酶能够访问基因组。资料设计：本实验室在标签随机六聚体聚合酶链式反应(T-PCR)的基础上，建立了一种对一个基因组当量(1-5FG)DNA敏感的全基因组扩增方法。这一创新来自于一种改进的引物设计，它稳定了聚合酶和引物-模板复合体。在这项提议中，基因组标记和扩增的两步方法将转化为使用薄层多孔聚丙烯酰胺水凝胶的两个兼容的固相聚合酶链式反应。为了从单个细胞中扩增出完整的基因组，将使用标准的微生物平板技术将大肠杆菌细胞空间分离到水凝胶表面，然后将细胞夹在两个反应层之间。全基因组扩增方法将进行优化，从单个分离的细胞中产生“测序准备”的DNA，片段大小由PCR延长时间控制。T-PCR方法产生的Polonies将从水凝胶中回收，并通过高通量454焦磷酸测序来确定基因组序列覆盖率和任何潜在的扩增偏向。对于第二阶段的研究，来自口腔-唾液微生物组的复杂微生物样本将使用固相Polony扩增进行评估。不同细胞类型的多样性需要增加细胞裂解/选择的步骤，紫外光催化将被探索作为一种手段来削弱微生物细胞壁，并提高在聚合酶链式反应过程中对热裂解的敏感性。以这种方式产生的Polonies将通过对16S rRNA基因进行测序来筛选，以评估恢复的整个基因组的微生物多样性。如果成功，这项技术将为空间分离和选择单个细胞进行全基因组扩增提供一种简单和容易获得的方法。 公共卫生相关性：我们将开发一种从水凝胶基质中的单个微生物细胞中培养、选择和扩增完整基因组DNA的元基因组学方法。利用固相聚合酶链式反应在空间上分离和扩增聚合酶克隆(Polonies)的能力将有助于扩大人类微生物组全基因组序列的参考库，从而更好地了解人类健康和疾病的微生物生态学。

DESCRIPTION (provided by applicant): The human microbiome represents a largely undefined consortium of organisms that may play a role in human health and disease. New technologies are needed to isolate and sequence individual "reference" genomes for a better understanding of the complex microbial ecology of the human host. Specific Aims: 1) To test a mechanism for isolating and amplifying polymerase colonies (polonies) from the whole genome of single cells using solid phase PCR in a polyacrylamide hydrogel; and 2) to explore UV-photocatalysis as a method of selectively weakening microbial cell walls, thereby rendering the genome accessible to DNA polymerase. Research Design: A whole genome amplification method that is sensitive to one genome equivalent (1-5 fg) of DNA was developed in this laboratory, based on tagged random hexamer PCR (T-PCR). The innovation comes from a modified primer design that stabilizes the polymerase and the primer-template complex. For this proposal, the two-step approach of genome tagging and amplification will be converted into two compatible solid-phase PCR reactions using thin layers of porous polyacrylamide hydrogel. In order to amplify whole genomes from single cells, standard microbiological plating techniques will be used to spatially isolate Escherichia coli cells onto the surface of the hydrogel and then sandwich the cells between the two reaction layers. The whole genome amplification method will be optimized for generating "sequencing-ready" DNA from individual isolated cells, with fragment size controlled by PCR extension time. Polonies generated by the T-PCR method will be recovered from the hydrogel and characterized by high throughput 454 Pyrosequencing to determine genome sequence coverage and any potential amplification biases. For the second phase of research, a complex microbial sample from the oral-salivary microbiome will be evaluated using solid-phase polony amplification. The potential for a diversity of different cell types requires an added step of cell lysis/selection, and UV photocatalysis will be explored as a means to weaken the microbial cell wall and improve susceptibility to heat lysis during PCR. Polonies generated in this manner will be screened by sequencing the 16S rRNA gene to assess microbial diversity of recovered whole genomes. Implications: If successful, this technology will provide a simple and readily accessible approach for spatially isolating and selecting single cells for whole genome amplification. PUBLIC HEALTH RELEVANCE: We will develop a metagenomics approach for plating, selecting, and amplifying whole genomic DNA from individual microbial cells in a hydrogel matrix. The ability to spatially isolate and amplify polymerase colonies (polonies) using solid-phase PCR will help expand the reference library of whole genome sequences from the human microbiome, leading to a better understanding of the microbial ecology of human health and disease.