DPCR for Viral Titrations and VCN: Rely on Sword Bio’s Unique Expertise

In molecular biology, Polymerase Chain Reaction (PCR) has revolutionized how we study and quantify nucleic acids. Two prominent techniques, Quantitative PCR (qPCR) and Digital PCR (dPCR), have emerged as powerful tools for sensitive and specific detection. While both technologies have their strengths, this blog post will focus on the advantages of dPCR over qPCR for viral titration and vector copy number assays.

qPCR measures the accumulation of DNA during a PCR reaction. The change in the intensity of the fluorescent signal measures the increase in the quantity of DNA at each cycle.  A comparison with a reference sample determines the number of original copies of template DNA in the reaction.

On the other hand, dPCR is a highly sensitive approach to nucleic acid detection and quantification. Each sample is partitioned into thousands of individual reactions, allowing for the detection of even the most negligible amounts of DNA. Each partition is analyzed after end-point PCR cycling for the presence or absence of a fluorescent signal, and the absolute number of molecules present in the sample is calculated.

1. dPCR for Viral Titration and Vector Copy Number Assays
   1. Increased Sensitivity and Precision
      1. dPCR can detect low levels of target molecules with high sensitivity, making it ideal for quantifying vector copy numbers in samples with low transgene expression. It distributes the sample across many minor microreactions, which helps reduce the variability in amplification efficiency and improves the precision and accuracy of quantification.
   2. Absolute Quantification
      1. Unlike qPCR, dPCR does not require a standard curve or reference to interpret overall quantity. This allows for accurate and sensitive quantification of the number of copies of a transgene or vector in a sample, providing valuable information about the stability and expression of the transgene.
   3. Differentiation Between Integrated and Episomal Vectors
      1. dPCR can differentiate between integrated and episomal vectors, allowing researchers to assess the stability and expression of the transgene over time.

In summary, while qPCR is helpful for high-throughput applications and where there are bigger differences in copy number, dPCR emerges as a superior choice for applications that require high sensitivity, precision, and absolute quantification. Its ability to detect small fold changes, such as a change in copy number from 5 to 6 copies, makes it highly suitable for viral titration and vector copy number assays that must be sensitive and very accurate.