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可能在人类健康和疾病中发挥作用的生物体的基本上不确定的财团。为了更好地了解人类宿主复杂的微生物生态学，需要新技术来分离和测序个体“参考”基因组。具体目标：1）测试在聚丙烯酰胺水凝胶中使用固相PCR从单细胞的全基因组分离和扩增聚合酶菌落（polonies）的机制;和2）探索UV-照射作为选择性削弱微生物细胞壁的方法，从而使基因组可接近DNA聚合酶。研究设计：本实验室基于标记随机六聚体PCR（T-PCR），建立了一种对1个基因组当量（1-5 fg）DNA敏感的全基因组扩增方法。这项创新来自于一种改良的引物设计，它可以稳定聚合酶和引物-模板复合物。对于这个提议，基因组标记和扩增的两步方法将被转换成两个兼容的固相PCR反应，使用多孔聚丙烯酰胺水凝胶薄层。为了从单细胞扩增全基因组，将使用标准微生物平板技术将大肠杆菌细胞空间分离到水凝胶表面上，然后将细胞夹在两个反应层之间。将优化全基因组扩增方法，以从单个分离细胞中产生“测序就绪”DNA，片段大小由PCR延伸时间控制。通过T-PCR方法产生的Polonies将从水凝胶中回收，并通过高通量454焦磷酸测序进行表征，以确定基因组序列覆盖率和任何潜在的扩增偏倚。对于研究的第二阶段，将使用固相聚合酶扩增来评估来自口腔-唾液微生物组的复杂微生物样本。不同细胞类型多样性的潜力需要增加细胞裂解/选择步骤，并且将探索UV照射作为削弱微生物细胞壁并提高PCR期间对热裂解的敏感性的手段。以这种方式产生的Polonies将通过16 S rRNA基因测序进行筛选，以评估回收的全基因组的微生物多样性。含义：如果成功，该技术将提供一种简单且易于获得的方法，用于空间分离和选择单细胞用于全基因组扩增。 公共卫生相关性：我们将开发一种宏基因组学方法，用于从水凝胶基质中的单个微生物细胞中接种，选择和扩增全基因组DNA。使用固相PCR在空间上分离和扩增聚合酶菌落（polonies）的能力将有助于扩大来自人类微生物组的全基因组序列的参考文库，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.