Selective microbial genomic DNA isolation using restriction endonucleases.

To improve the metagenomic analysis of complex microbiomes, we have repurposed restriction endonucleases as methyl specific DNA binding proteins. As an example, we use DpnI immobilized on magnetic beads. The ten minute extraction technique allows specific binding of genomes containing the DpnI Gm6ATC motif common in the genomic DNA of many bacteria including γ-proteobacteria. Using synthetic genome mixtures, we demonstrate 80% recovery of Escherichia coli genomic DNA even when only femtogram quantities are spiked into 10 µg of human DNA background. Binding is very specific with less than 0.5% of human DNA bound. Next Generation Sequencing of input and enriched synthetic mixtures results in over 100-fold enrichment of target genomes relative to human and plant DNA. We also show comparable enrichment when sequencing complex microbiomes such as those from creek water and human saliva. The technique can be broadened to other restriction enzymes allowing for the selective enrichment of trace and unculturable organisms from complex microbiomes and the stratification of organisms according to restriction enzyme enrichment.

Circulating tumor cells are transcriptionally similar to the primary tumor in a murine prostate model.

The abundance of circulating tumor cells (CTC) indicates patient prognosis. Molecular characterization of CTCs may add additional information about a patient's disease. However, currently available methods are limited by contamination with blood cells. We describe a study using a modified CTC-chip to capture CTCs from an orthotopic xenograft model. Using laser capture microscopy to collect CTCs from the chip, we compared transcripts from purified CTCs with those from primary and metastatic tissue. Transcriptional profiles showed strong concordance among primary, metastatic, and CTC sources. Moreover, cells captured on the chip were viable and could be expanded in culture. We conclude that the CTC-chip is a useful tool to further characterize animal models of cancer and that viable CTCs can be isolated and show transcriptional similarity to solid tumors.