Rapid Recovery of DNA from Dried Blood Spots: Use of a Novel Electrophoretic Method

Abstract

The demand for rapid, effective point of care diagnostics has created a pressing need for new and improved methods to quantify DNA from patient samples. Dried blood spotting is a well‐established testing methodology that has gained increasing importance in in vitro diagnostics due to its low cost, low sampling requirements, and long‐term sample stability. In recent years, dried blood spots have been increasingly used for targeted genetic and metabolite profiling. Large‐scale genetic screening of neonatal dried blood spots has a great potential to lower patient mortality and morbidity through early diagnosis of primary immunodeficiencies. However, DNA extraction from the surface of the dried blood spot remains the most time consuming, costly and labor‐intensive part of DNA analysis. Thus, the objective of this study was to develop and optimize a simple and rapid methodology to extract DNA from dried blood spots.In the present study, we created a novel methodology to extract DNA from a dried blood spot using only a lysis buffer (10mM KOH/30mM Tris buffer), 50V, and heat. Briefly, during the electric field extraction spots and lysis buffer were placed in an electroporation cuvette and DNA was extracted by applying 25–50 volts across the plates of the cuvette. The spot was then removed and DNA in the supernatant was analyzed using qPCR or fluorescent nucleic acid stains. This methodology was optimized by the addition of a 70°C heating step before the electric field extraction to better retrieve episomal DNA.This electric field DNA extraction is to our knowledge, the first methodology to use an electric field to extract episomal DNA from a dried blood spot. Results of this study show that this methodology produces significant recoveries of both genomic and episomal DNA (Ct (FAM) 33.0 (50V, 60s) to Ct (FAM) 35.0 (heating alone) p <0.01). Varying recoveries of episomal and genomic DNA were also seen at different voltage conditions suggesting that this methodology may be useful for the isolation and purification of episomal DNA (FAM (25V, 300s (no amplification) and 50V, 60s (Ct 33.0)). Furthermore, preliminary qualitative and spectroscopic analyses suggest that protein removal could be achieved using this dual voltage technique. The 25‐minute electric field extraction procedure has one of the lowest times for the extraction of DNA found within the literature. This optimized procedure negates the need for costly treatment and wash steps, simplifying DNA extraction considerably. Most importantly, the electric field extraction reduces the time of proprietary procedures by more than 30 minutes, while retaining 75–80% of the yield. Thus, the results show that this novel method of electric field extraction provides a rapid and effective protocol to remove episomal and genomic DNA from dried blood spots. This optimized electric field device not only simplifies bioanalytical techniques, but provides a significant step towards point of care diagnostics and should be expanded to other sample types.Support or Funding InformationThe authors thank PerkinElmer for providing the funding for this study.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.