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大部分未定义的生物体联合体。为了更好地了解人类宿主复杂的微生物生态，需要新的技术来分离和测序个体“参考”基因组。具体目的：1)在聚丙烯酰胺水凝胶中利用固相PCR从单细胞全基因组中分离和扩增聚合酶菌落（polonies）的机制；2)探索紫外线光催化作为一种选择性削弱微生物细胞壁的方法，从而使DNA聚合酶能够接近基因组。研究设计：基于标记随机六聚体PCR (T-PCR)，本实验室开发了一种对一个基因组当量（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，它稳定了聚合酶和引物-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转化为两个兼容的固相PCR反应，使用薄层多孔聚丙烯酰胺水凝胶。为了从单细胞中扩增全基因组，将使用标准微生物电镀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。将优化全基因组扩增方法，从单个分离细胞中产生“测序就绪”的DNA，片段大小由PCR延长时间控制。由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差。对于第二阶段的研究，将使用固相极化扩增技术对口腔-唾液微生物组的复杂微生物样本进行评估。不同细胞类型多样性的潜力需要一个额外的细胞裂解/选择步骤，紫外光催化将被探索作为一种手段，在PCR过程中削弱微生物细胞壁，提高对热裂解的敏感性。通过对16S rRNA基因进行测序来筛选这种方式产生的poloni，以评估恢复的全基因组的微生物多样性。意义：如果成功，该技术将为空间分离和选择单细胞进行全基因组扩增提供一种简单易行的方法。

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.