Abstract
We have shown that DNA can be extended on glass slides permitting the observation of fluorescently labeled DNA fibers and facilitating the quantitation of their length. When the stretched DNA fibers are also immunostained for the presence of apurinic/apyrimidinic (AP) sites, it was possible to quantitate the number of AP sites per unit length of DNA. Measurements of AP site frequency that are determined in this manner are comparable in accuracy to the “gold standard” method of AP site quantitation, but have a lower variance and the ability to analyze even single DNA molecules. Using this method, it has been possible to detect the increased frequency of AP site formation in DNA of cells exposed to hydrogen peroxide in cell culture. Furthermore, it was possible to show that DNA located at replication sites were more intensely damaged than the remainder of the DNA. Similar increases in the quantity of DNA damage sites have also been found in the DNA of cells treated with the cancer chemotherapeutic drug doxycycline. While this technique appears to be a powerful method to detect DNA damage in cells exposed to damaging agents in culture and in vivo, it has the liability of being extremely labor intensive and requires an extended time (weeks to days) for completion. To remedy these shortcomings and to allow clinical translation of this technology, we have developed an alternate approach, which involves the extraction of DNA from circulating tumor cells isolated from whole blood using a unique microfluidic device, immunostaining the DNA and the associated DNA AP sites in micro‐scale reaction chambers and the alignment and deposition of individual DNA fibers in nanochannels (<100 nm in width and depth). This enables single DNA molecules to be stretched reproducibly in nanochannels to near their full contour length which facilitates quantification of labeled AP sites. The DNA fibers positioned within the nanochannels are fluorescently imaged and the images subjected to a unique image analysis technique to provide highly quantitative information on AP site frequency. When using this image analysis approach, it is possible to obtain statistically significant results from even single cells. The combination of these microfluidic and nanofluidic devices into a single analysis platform for quantitating rates of DNA damage reduces the time required for this measurement to an hour or less, reduces technical time to that needed to load samples and reagents, and should permit these analyses to be made in the clinical setting by clinical laboratory personnel.Support or Funding InformationSupported by NIH Grants R21‐125337 and P41‐EB020594.
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