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Quantification of Adeno-Associated Viral Genomes in Purified Vector Samples by Digital Droplet Polymerase Chain Reaction
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Evaluation of digital PCR for absolute DNA quantification.

Rebecca Sanders1, Jim F Huggett, Claire A Bushell

  • 1LGC, Queens Road, Teddington, Middlesex TW11 0LY.

Analytical Chemistry
|March 31, 2011
PubMed
Summary

Microfluidic digital PCR (dPCR) accurately quantifies low DNA levels, showing high reproducibility and sensitivity. However, template type, preamplification, and measurement methods can introduce bias in digital PCR assays.

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Analytical Chemistry

Background:

  • Microfluidic digital PCR (dPCR) is an emerging technique for nucleic acid quantification.
  • It shows promise for sensitive detection of low-level targets.

Purpose of the Study:

  • To evaluate the quantitative performance of dPCR for measuring small DNA amounts (<200 copies).
  • To investigate factors influencing dPCR's technical performance, including precision, sensitivity, and reproducibility.

Main Methods:

  • Utilized various DNA templates, matrices, and primer sets to assess dPCR performance.
  • Evaluated reproducibility across different times and primer sets targeting the same molecule.
  • Assessed the impact of carrier presence and preamplification on measurement accuracy.

Main Results:

  • dPCR demonstrated high reproducibility and good analytical sensitivity, even outside manufacturer-recommended ranges.
  • Measurement precision improved with carrier inclusion, though quantity estimates were unaffected.
  • Discrepancies were observed between different template types and between dPCR and UV spectrophotometry.
  • Preamplification introduced significant measurement bias.

Conclusions:

  • dPCR is a reproducible technique for low copy number DNA quantification.
  • Experimental design factors such as template type, carrier presence, and preamplification significantly impact dPCR accuracy.
  • Careful consideration of these factors is crucial for reliable dPCR results.