Single cell trapping and DNA damage analysis using microwell arrays

Abstract

With a direct link to cancer, aging, and heritable diseases as well as a critical role in cancer treatment, the importance of DNA damage is well-established. The intense interest in DNA damage in applications ranging from epidemiology to drug development drives an urgent need for robust, high throughput, and inexpensive tools for objective, quantitative DNA damage analysis. We have developed a simple method for high throughput DNA damage measurements that provides information on multiple lesions and pathways. Our method utilizes single cells captured by gravity into a microwell array with DNA damage revealed morphologically by gel electrophoresis. Spatial encoding enables simultaneous assays of multiple experimental conditions performed in parallel with fully automated analysis. This method also enables novel functionalities, including multiplexed labeling for parallel single cell assays, as well as DNA damage measurement in cell aggregates. We have also developed 24-and 96-well versions, which are applicable to high throughput screening. Using this platform, we have quantified DNA repair capacities of individuals with different genetic backgrounds, and compared the efficacy of potential cancer chemotherapeutics as inhibitors of a critical DNA repair enzyme, human AP endonuclease. This platform enables high throughput assessment of multiple DNA repair pathways and subpathways in parallel, thus enabling new strategies for drug discovery, genotoxicity testing, and environmental health.base excision repair | comet assay | DNA repair D NA damage is a critical risk factor for cancer, aging, and heritable diseases (1-3), and it is the underlying basis for most frontline cancer therapies (4). Increased knowledge about DNA damage and repair would facilitate disease prevention, identification of individuals at increased risk of cancer, discovery of novel therapeutics, and better drug safety testing. Despite their obvious translational impact, most DNA damage assays have not changed significantly in more than two decades, and thus are not compatible with modern high throughput screening technologies. To better assess the biological impact of damage, we need data that reflect the integrated effect of multiple DNA repair pathways and subpathways, in response to a range of DNA lesions, measured in an accurate and high throughput fashion.The single cell gel electrophoresis or "comet" assay is based on the principle that relaxed loops (induced by single strand breaks) and DNA fragments migrate farther in an agarose gel than undamaged DNA (5-8). The alkali comet assay sensitively detects a range of DNA lesions, including strand breaks (single and double), as well as alkali sensitive sites. Although most base lesions are not directly detected, base adducts can be detected when converted to abasic sites or single strand breaks with the addition of purified DNA repair enzymes (7-11). The comet assay has been used in a variety of applications, including genotoxicity testing, human biomonitoring and epidemiology, environmental healt...

Multiplexed DNA repair assays for multiple lesions and multiple doses via transcription inhibition and transcriptional mutagenesis

“…Lymphoblastoid WT None GM15510 (8) Lymphoblastoid WT None GM15213 (9) Lymphoblastoid WT None GM15221 (10) Lymphoblastoid WT None GM15227 (11) Lymphoblastoid WT None GM15385 (12) Lymphoblastoid WT None GM15590 (13) Lymphoblastoid WT None GM15038 (14) Lymphoblastoid WT None GM15056 (15) Lymphoblastoid WT None GM15072 (16) Lymphoblastoid WT None GM15144 (17) Lymphoblastoid WT None GM15216 (18) Lymphoblastoid WT None GM15226 (19) Lymphoblastoid WT None GM15242 (20) Lymphoblastoid WT None GM15268 (21) Lymphoblastoid WT None GM15324 (22) Lymphoblastoid WT None GM15386 (23) Lymphoblastoid WT None GM15061 (24) Lymphoblastoid WT None…”

“…Measuring the consequences of DNA repair in intact cells by monitoring sister chromatid exchanges, chromosome aberrations, or DNA strand breaks by comet assays also integrates complexity into a single readout, but it requires labor-intensive analyses that make it refractory to implementation in a clinical setting (21)(22)(23). Although recently developed high-throughput comet assays provide an excellent alternative, they are limited to DNA damage that leads to, or can be converted to, DNA strand breaks (24).…”

Block-Cell-Printing for live single-cell printing

“…Several approaches have been designed for this purpose, e.g., inkjet cell printing (3)(4)(5)(6), surface engineering (7)(8)(9)(10)(11)(12)(13)(14)(15), and physical constraints (16)(17)(18)(19)(20)(21)(22)(23). However, finding a method that completely satisfies the above requirements remains a challenge.…”

High throughput toxicity screening and intracellular detection of nanomaterials

“…The ‘CometChip’ integrates a HTS comet assay with automated scoring in a novel way; cells are deposited at predefined positions stamped in a microarray on an agarose‐coated plate, so that it is possible to locate comets precisely for image capture and analysis 131, 132…”