Abridged solid-phase extraction with alkaline Poly(ethylene) glycol lysis (ASAP) for direct DNA amplification

Area of Science:

• Molecular Biology
• Biotechnology
• Analytical Chemistry

Background:

• Sample preparation is a bottleneck for point-of-need nucleic acid amplification testing.
• Current methods for DNA extraction are often complex and time-consuming.
• Developing rapid and simple sample preparation is crucial for advancing sample-in-answer-out devices.

Purpose of the Study:

• To develop a consolidated, rapid, and simple sample preparation method for nucleic acid amplification testing.
• To integrate alkaline lysis and solid-phase extraction for direct on-bead amplification.
• To improve the efficiency and sensitivity of DNA extraction for point-of-need applications.

Main Methods:

• Consolidation of alkaline poly(ethylene) glycol (PEG)-based lysis and solid-phase extraction using a single reagent.
• Optimized reagent composition: 15% PEG8000, 0.5 M NaCl, and 3.5 mM KOH.
• Direct on-bead amplification with 1.5 μg beads per 20 μL PCR reaction mix.

Main Results:

• A novel 5-minute method significantly improved detection limits (10-100 fold) compared to commercial kits and previous methods.
• Sensitivity was further enhanced by an ethanol wash or extended incubation, enabling single-copy detection of Escherichia coli DNA.
• The method showed comparable performance in biological fluids (saliva, sweat, urine) with a minor delay in the 5-minute protocol.

Conclusions:

• The proposed consolidated method offers a practical and chemical approach for simplified DNA sample preparation.
• This technique is highly attractive for integration into point-of-need testing workflows.
• The method streamlines sample preparation, accelerating the development of sample-in-answer-out devices for nucleic acid amplification testing.