Indirect purification method provides high yield and quality ssDNA sublibrary for potential aptamer selection

Area of Science:

• Molecular Biology
• Biochemistry
• Biotechnology

Background:

• High-quality single-stranded DNA (ssDNA) is crucial for aptamer selection.
• Existing ssDNA purification methods suffer from low yield or structural damage.
• Damage to ssDNA tertiary structure can impede subsequent aptamer selection processes.

Purpose of the Study:

• To develop an improved method for generating and purifying ssDNA sublibraries.
• To achieve high yield and preserve the structural integrity of ssDNA.
• To provide a ssDNA purification strategy suitable for aptamer SELEX (Systematic Evolution of Ligands by EXponential enrichment).

Main Methods:

• Employed symmetric PCR to generate sufficient DNA templates.
• Utilized asymmetric PCR with specific primer concentrations (excess non-biotinylated forward, insufficient biotinylated reverse).
• Incorporated a biotin-streptavidin system for efficient dsDNA elimination without harsh chemical treatments.

Main Results:

• The indirect purification method successfully generated a high-yield ssDNA sublibrary.
• Agarose gel electrophoresis confirmed the absence of dsDNA and by-product contamination.
• Achieved a significantly higher purified ssDNA concentration (1020±210nM) compared to previous methods.

Conclusions:

• The novel indirect purification method offers a simple and rapid approach for obtaining high-quality ssDNA.
• This technique effectively preserves ssDNA tertiary structure, essential for aptamer selection.
• The method overcomes the trade-offs between yield and quality seen in conventional ssDNA purification strategies.