Development of DNA Extraction Method from Dried Blood Spots for Genetic Analysis
Keywords:
Dried blood spot (DBS), DNA extraction, Magnetic beads, Next generation sequencingAbstract
Genetic analysis using Next Generation Sequencing (NGS) technology is an important tool in medical genetics, enabling precise and comprehensive diagnosis of genetic disorders. It has played an increasingly significant role in newborn screening. This study aimed to develop a DNA extraction method from dried blood spots (DBS) to obtain DNA with sufficient quality and quantity for the detection of hearing loss using Targeted Next Generation Sequencing. Three extraction methods were compared: a commercial extraction kit, a magnetic bead-based method combined with an in-house lysis buffer, and a magnetic bead-based method with a commercial lysis buffer. The results showed that DNA extracted from all three methods had an average concentration of 5.28–6.88 ng/µL, purity ratios (A260/A280) ranging from 1.97–2.08, and (A260/A230) ranging from 0.2–1.33. DNA integrity numbers (DIN) were between 8.8–9.9. PCR amplification using both singleplex and multiplex assays produced target fragments of 233–570 bp as expected. NGS data generated by the DNA Nanoball Sequencing (DNBSEQ-E25) platform from MGI demonstrated high sequencing quality, with mapping quality of 99.85–99.87%, 30× coverage of 97.13–99.25%, duplicate rate of 7.87–9.11%, and base call accuracy (Q30) of 90.21–90.72%. This study demonstrates that the magnetic bead-based extraction method combined with an in-house lysis buffer provides DNA yield and quality comparable to the commercial extraction kit, and is suitable for NGS-based genetic analysis for hearing loss screening.
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