Evaluating digital PCR for the quantification of human nuclear DNA: determining target strandedness

Area of Science:

• Molecular Biology
• Analytical Chemistry
• Metrology

Background:

• Digital polymerase chain reaction (dPCR) is a powerful tool for DNA concentration measurement.
• Potential bias in dPCR arises from single-stranded DNA (ssDNA) in double-stranded DNA (dsDNA) samples.
• Accurate quantification of ssDNA is essential for realizing the metrological potential of dPCR.

Purpose of the Study:

• To evaluate methods for determining the proportion of ssDNA in dsDNA samples for dPCR.
• To identify reliable analytical approaches for ssDNA quantification in dPCR workflows.

Main Methods:

• Investigated A260 ratio, dPCR ratio, cdPCR staircase, and ddPCR endonuclease assays.
• Focused on endonuclease-based methods for accurate ssDNA proportion estimation.
• Validated enzyme-assay pairs using human nuclear DNA extracts.

Main Results:

• Endonuclease-based approaches provided the most accurate ssDNA proportion estimates.
• Significant variability (up to 50%) in ssDNA content was observed between different assays.
• Commercial PCR calibrants showed higher ssDNA content (≈18%) compared to extracted DNA (≤6%).

Conclusions:

• Endonuclease-based assays are recommended for accurate ssDNA quantification in dPCR.
• Assay-specific differences in ssDNA measurement may relate to fragment guanine-cytosine (GC) content.
• Careful consideration of ssDNA content is vital for high-accuracy dPCR applications.