Monitoring long-term DNA storage via absolute copy number quantification by ddPCR.

Clicks: 191
ID: 34174
2019
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Abstract
Long-term storage of DNA is a routine practice in biomedical research, diagnostics and drug discovery. Periodic monitoring is important for early detection of changes in DNA quality and quantity. Existing methods include agarose gel, ultraviolet (UV) absorbance, fluorometric reading and qPCR. However, these methods are either limited by sensitivity or depend on DNA standards, which face the same storage challenges. In this paper, we tested the state-of-the-art droplet digital PCR (ddPCR) technology that can quantify the absolute DNA copy number with no need of a standard curve. We found that ddPCR was very accurate in determining the level of a plasmid DNA standard and was sensitive to DNA loss due to degradation or adsorption. With the ddPCR technology, we found a gradual process of DNA adsorption to several types of low binding tubes, which was unnoticed before. Although modest, adsorption significantly affected recovery of highly diluted DNA (<0.2 μg/mL), which could be rescued by addition of carrier DNA. In conclusion, this paper not only demonstrated that ddPCR is an ideal method for monitoring DNA storage, but also provided an effective approach to improving recovery of highly diluted DNA, which may have broad implications in assay development, diagnostics and forensic sciences.
Reference Key
wang2019monitoringanalytical Use this key to autocite in the manuscript while using SciMatic Manuscript Manager or Thesis Manager
Authors Wang, Yu;Keith, Marie;Leyme, Anthony;Bergelson, Svetlana;Feschenko, Marina;
Journal analytical biochemistry
Year 2019
DOI
S0003-2697(19)30556-1
URL
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