DNA Amplification of the Whole Genome from a Single Cell

70555 Understanding the contributions of different bacterial strains or species in a complex bacterial community requires multiple experimental approaches to determine the biochemistry of each community member. One such approach involves the determinination of the complete DNA sequence of each prokaryote cell, a process that is difficult and time-consuming with culturable organisms, and currently impossible with unculturable cells. This project will refine the protocols for cell lysis and DNA amplification, using a multiple displacement amplification method (MDA) to generate sufficient highly-representative DNA from single unculturable microorganisms, in order to produce complete genome sequences from the amplified DNAs. In Phase I, a model bacterial system, composed of non-pathogenic strains of gram positive and gram negative culturable bacteria, with and without plasmids, will be used to optimize MDA for amplification of the whole genome from a single cell. This DNA amplification method will be validated by demonstrating uniform amplification of all loci of a single prokaryotic cell in a mixed bacterial community. Commercial Applications and Other Benefits as described by the awardee: The technology should contribute substantially to the DOE Genomes to Life Project by facilitating the sequencing of unculturable organisms in bacterial communities, and also aid in developing a robust protocol for the amplification of DNA from a single eukaryotic cell. Biomedical applications include the diagnosis of genetic disease from early embryos, nucleated erythrocytes from maternal blood, genetic recombination analysis from a single sperm, and mutation analysis of single tumor cells.