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.
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.
|Bigdeli, Saharnaz; Dettloff, Roger O; Frank, Curtis W et al. (2015) A simple method for encapsulating single cells in alginate microspheres allows for direct PCR and whole genome amplification. PLoS One 10:e0117738|