Nucleic acid extraction from complex biofluid using toothpick-actuated over-the-counter medical-grade cotton

Area of Science:

• Biomaterials Science
• Molecular Diagnostics
• Biotechnology

Background:

• Nucleic acid amplification techniques (NAATs) like qPCR are crucial for molecular diagnostics but require pure nucleic acids.
• Traditional DNA extraction methods often necessitate specialized equipment like centrifuges, limiting their use in resource-constrained settings.
• Developing simple, instrument-free DNA extraction methods is essential for expanding NAAT accessibility.

Purpose of the Study:

• To develop and evaluate an instrument-free method for genomic DNA (gDNA) extraction using readily available materials.
• To assess the feasibility of using medical-grade cotton for gDNA extraction from complex biological samples.
• To determine the efficiency and quality of DNA extracted using this novel method for downstream molecular applications.

Main Methods:

• Screened medical-grade cotton as a biomaterial for DNA extraction.
• Performed instrument-free extraction of gDNA spiked in varying concentrations of serum or cell lysate using cotton and a toothpick.
• Utilized chaotropic salts optionally to compare extraction times and efficiency.
• Validated extracted DNA quality using Polymerase Chain Reaction (PCR), agarose gel electrophoresis, quantitative PCR (qPCR), and Sanger sequencing.

Main Results:

• Successfully extracted gDNA from samples containing 30-60% serum or cell lysate in an instrument-free manner.
• Extraction times were as short as 10 minutes (without chaotropic salt) or 30 minutes (with chaotropic salt).
• Extraction efficiency ranged from 0.3% to 27%, influenced by sample composition.
• Extracted DNA was suitable for PCR amplification and Sanger sequencing, indicating feasibility for NAATs.

Conclusions:

• Medical-grade cotton can be effectively used for instrument-free DNA extraction with a simple toothpick.
• This method offers a low-cost, accessible alternative for DNA purification in resource-limited settings.
• The extracted DNA quality is sufficient for downstream molecular diagnostic applications, enhancing NAAT accessibility.