Massachusetts Institute of Technology
Comet-Chip: A High-Throughput DNA Damage Sensor for Environmental Health Studies
Bevin P. Engelward
DNA damage is an important risk factor for cancer and many other diseases. Whether induced by the environment or created endogenously, covalent modifications to DNA structure can be both cytotoxic and mutagenic. Being able to measure DNA damage and repair in human samples is, therefore, fundamentally valuable, both for delineating environmental conditions that render cells vulnerable to mutations, and for revealing genetic factors that modulate susceptibility to DNA damage.
The single cell gel electrophoresis assay, or 'comet assay' is one of the most sensitive and versatile approaches for measuring DNA damage in human cells. It is grounded on a simple principle: when visualizing electrophoresed cells embedded in agarose, undamaged DNA is supercoiled and highly compact, whereas damaged DNA (relaxed loops and fragments) can more readily migrate, giving rise to the appearance of a bright nucleoid with a comet-like tail. Despite its proven efficacy, the comet assay is underutilized in studies of environmental risk factors in epidemiological studies, mostly because of a lack of standardization that has led to inconsistent results among researchers as well as the time/labor required to perform the assay.
With support of the NIEHS, we have applied lab-on-a-chip technologies to create a "CometChip." To achieve the desired reliability and throughput, we have exploited microarray technology to create a single cell array that can then be processed for analysis of DNA damage. Using microfabrication technology, a mask with openings of as little as 20 um in diameter and as much as 50 um diameter is used to create a surface of arrayed micropegs that are then submerged into molten agarose to create arrayed microwells. Wells can be designed to accommodate single cells or multiple cells.
After capture, cells can be cultured on-chip, exposed to DNA damaging agents, and assayed at various times post exposure. By fixing samples at various times post-exposure, repair kinetics can quantified with unprecedented efficiency. By applying and optimizing existing techniques for analysis of different types of DNA lesions, we have created conditions wherein the kinetics of base excision repair of oxidative damage, base excision repair of methylation damage, nucleotide excision repair of UV dimers, unhooking of DNA crosslinks, and non-homologous end joining of double strand breaks can all be quantified in a high throughput fashion.
Importantly, the platform has utility both with low-tech microscopy platforms and high throughput automated imaging stations. We have also exploited the throughput of this system to evaluate DNA repair kinetics among ethnically diverse individuals. We anticipate this technology will render this assay useful in a broad range of clinical, epidemiological, and experimental settings. Such a high-throughput DNA damage and response sensor will be invaluable both for discerning dangerous environmental exposures and evaluating the efficacy of policy decisions aimed at reducing relevant exposures and preventing cancer and other health effects related to DNA damage.
- Ability to analyze dozens of samples in parallel; one month's worth of data collection can now be collected in ~2 days
- Suppression of sample-to-sample variation
- Ability to load cells at concentration that range over two logs (thus obviating the need for precise loading concentrations)
- Prevention of comet tail overlap that often prevents analysis using the traditional method
- Ability to analyze multiple DNA repair pathways
- Validated efficacy for detecting specific repair pathway activity, including BER, NER and NHEJ
- Methods for multiplexing enable analysis of multiple end points in a single cell
- Effective for quantification of DNA damage in clusters of multiple cells
- Useful for a broad range of cell types
- Cells can be cultured on the same platform that they are treated and analyzed in
- Ligands can be incorporated into the gel to enable culturing and analysis of contact-dependent cell types
- High throughput automated imaging and analysis permits data collection for hundreds of samples (with hundreds of cells each) in under a day