DNA Concentration from PCR: Key Measurements and Factors for Accurate Results

Understanding DNA Concentration from PCR

DNA concentration from PCR measures the amount of double-stranded DNA generated during amplification, but accurate quantification requires verifying product size to avoid misleading results. PCR amplifies target DNA, producing various by-products affecting concentration measures.

Measuring DNA Concentration

The Qubit fluorometer provides a direct measurement of the total double-stranded DNA concentration post-PCR. It uses fluorescent dyes that selectively bind to double-stranded DNA, giving a precise quantification. However, Qubit cannot distinguish between specific amplicons and non-specific products such as primer dimers.

Verifying PCR Product Size

Accurate interpretation of DNA concentration depends on confirming the PCR product size. Techniques like gel electrophoresis, Bioanalyzer, or Tapestation visualize the size distribution of DNA fragments.

• Gel electrophoresis separates DNA by size, allowing direct visualization of expected bands.
• Bioanalyzer and Tapestation provide higher resolution size profiles and quantitation curves.

This step ensures that observed DNA concentrations represent the desired amplicon rather than artifacts.

Avoiding Overestimation from Primer Dimers

Primer dimers are short non-specific products formed by primers binding to each other, which can inflate DNA concentration measurements. For example, a Qubit reading may suggest hundreds of ng/μL of DNA when much is primer dimers. Checking size distribution prevents overestimation by detecting these small unwanted products.

Factors Affecting PCR Yield

The amount of PCR product depends on the initial microbial load in the sample. Samples with low template quantity may yield less DNA despite the same cycle number. Increasing PCR cycles beyond 12 typically amplifies products but can increase non-specific by-products and primer dimers.

Optimizing the number of cycles balances yield and specificity, ensuring reliable DNA concentrations suitable for downstream applications.

Key Takeaways

• Qubit measures total double-stranded DNA but cannot discern specific PCR products.
• Visualizing PCR products by gel or Bioanalyzer confirms expected amplicon size.
• Primer dimers can cause significant overestimation of DNA concentration.
• PCR product concentration varies with sample microbial load and cycle number.