Thermostable primer-free whole genome amplification of single cells with viral en

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DESCRIPTION (provided by applicant): The genetic basis of disease remains a significant research problem, impacting the fields of cancer biology, neurology, cardiology, development, and even microbiology, as recently realized by the Human Microbiome Project. Determination of the genomic sequence underlying observed phenotypes is now widespread. Genomic sequencing is becoming faster and cheaper, allowing access to more genomic information and greater understanding of the relationships between genotype and phenotype. Increasingly, these technologies are being applied to smaller and smaller sample sizes, down to single cells. A single cell contains all the necessary genetic information, and theoretically can be amplified repeatedly. However, generating enough amplified DNA from such small samples for genomic sequencing is constrained by several features of current technologies. Current amplification protocols use phi29 polymerase and DNA primed with random hexamers. Although this system has allowed for the study of disease using single cells, it has numerous disadvantages. Primary among these are primer amplification artifacts and high bias which is especially exacerbated when the amount of target DNA is low. Biased amplification from single genomes results in loss of sequence information such as allelic dropout and complicates copy number variant analysis. This bias is stochastic because it results from the initial annealing of random hexamers to begin amplification from random regions. Such stochastic bias makes comparison between single cells nearly impossible. The goal of this Phase I proposal is to enable primer-free DNA synthesis, to vastly improve the efficiency and coverage of amplified DNA from samples as small as single cells. Removing primers will eliminate unproductive side-reactions and decrease bias in the final product. This system is expected to operate isothermally at higher temperatures, without the need for an initial denaturation step. The commercial result of our proposed study will be a reagent kit for high fidelity, low bias (high coverage) amplification of DNA from single cells without primer background issues. This work will advance the study of single cell genomics by providing higher sequence coverage, enabling research into cancer, stem cells, biofilms, and the human microbiome.