Dna Sequencing Of Uncultured Microbes From Single Cells

Development of a method to sequence DNA from a single cell has enabled new strategies to investigate the microbial world. Only a few years ago, sequencing from one cell was not feasible. A bacterium only contains a few femtograms of DNA which is insufficient for current sequencing technologies. This limitation was overcome with the development of a method to amplify DNA called multiple displacement amplification (MDA) which can generate micrograms of genomic sequence from one cell. Improvements have also been made in our ability to isolate cells by flow cytometry, micromanipulation and microfluidics and to lyse the cells to release the single genome copy as a template for MDA. Large pori:ions of the genome can be obtained from each cell and this has opened up a new front in the effort to sequence uncultivated species. Cells can be isolated from an environment or clinical specimen and directly sequenced with no need to develop culture methods. This chapter will review the current methodologies, the strengths and limitations of the single-cell approach and its application to microbial genomics.