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BioSpotlight
 
Patrick C. H. Lo, Ph.D. and Kristie Nybo, Ph.D.
BioTechniques, Vol. 55, No. 3, September 2013, p. 101
Full Text (PDF)

More mitochondrial DNA

Mitochondrial DNA (mtDNA) suffers a higher rate of mutation compared to nuclear DNA, due to factors including proximity to highly reactive oxygen species, lack of histone protection, and poor fidelity of mtDNA replication and repair. The result of this higher mutation rate is heterogeneity within the mtDNA population of a cell, a useful indicator of the cumulative effects of endogenous and environmental DNA damaging agents, as well as general cellular deterioration. Next-generation sequencing (NGS) is widely used to catalogue and analyze mtDNA sequence heterogeneity. However, even with multiple copies of mtDNA in each cell, mtDNA represents only a small fraction of the total DNA, necessitating enrichment schemes to isolate mtDNA prior to NGS. Enrichment techniques such as ultracentrifugation on CsCl gradients require special instrumentation, while PCR amplification of mtDNA from total DNA samples can potentially lead to unwanted amplification artifacts. As a simpler, faster, and more affordable alternative, A.Y. Maslov and his group at the Albert Einstein College of Medicine (New York, NY) describe a new method for preparing samples of highly enriched mtDNA suitable for direct sequencing. The method is composed of two steps: (i) mtDNA is isolated using a standard bacterial plasmid DNA miniprep kit, and (ii) the isolated DNA is purified by reversible binding on solid-phase paramagnetic beads. The use of DNA miniprep kits for mtDNA isolation is based the similarity of mtDNA size to that of plasmid DNA, a fact exploited in other mtDNA isolation techniques. However, when followed by the bead purification steo recommended by Maslov and his colleagues, the level of mtDNA purification, as measured by qPCR, is greatly enhanced, with a ~2000-fold enrichment relative to total cellular DNA, compared to a ~10-fold enrichment for 2 commercial mtDNA kits tested. mtDNA purified with this new method performed well in next-generation sequencing, resulting in 22% and 99% of reads aligning to mtDNA for unamplified samples and for samples enriched by 10 PCR cycles, respectively. While this new method is technically simple and rapid, it is the high level of enrichment that will truly help scientists with their studies of mtDNA.

See “Fast mitochondrial DNA isolation from mammalian cells for next-generation sequencing”.

Engulfed

Phagocytosis occurs when dedicated phagocytic cells, such as macrophages, recognize a target object or pathogen and respond by recruiting organelles to internalize it. Since the process is essential in development and innate immune response, demand for experimental procedures to accurately assay phagocytosis in a high-throughput fashion is increasing. While current approaches take advantage of flow cytometry or image analysis to detect particles residing either inside or outside the cel l, no assay has yet been developed to detect the intermediate stages of phagocytosis. To address this need, Yeo et al. at the University of Queensland (Brisbane, Australia) developed an image-based, semi-automated assay to quantify engulfment of opsonized beads in three stages of phagocytosis: (i) beads attached to the cell surface, (ii) beads partially engulfed by phagocytic cups, and (iii) fully internalized beads. To do this, the authors applied latex beads bound with human IgG to cells. After a specified time, the cells were fixed and human IgG detected using fluorescent antibodies. Microscopy was used to determine the total number of beads in a visual field and then identify the stage of engulfment for each bead by assessing the percentage of visible fluorescent label on that bead. Automated bead counts and identification of phagocytic stages proved highly reproducible, matching well with manual counts across all three stages of phagocytosis. A time course study demonstrated that changes in internalization were accurately assessed, which also benefited studies using drugs to impede early stages of phagocytosis. Taken together, these experiments demonstrate that this new assay is sensitive, reproducible, and unbiased. And by using automated image analysis, this approach also becomes amenable to high-throughput screening, with the potential to facilitate large-scale studies on the effects of drugs, small molecules, or RNAi on phagocytosis.



See “High-throughput quantification of early stages of phagocytosis”.