Digital PCR Instructional Module

Introduction

• Speaker: Frank Biswarn, Digital Biology Group, BioRad Laboratories
• Focus: Introduction to digital PCR (dPCR) and accurate counting of nucleic acid molecules.

Key Concepts

• Quantifying Nucleic Acids:
  • Common question in molecular biology: "How many copies of my target molecule are present?"
  • Common technique: Quantitative PCR (qPCR).
  • Limitations of qPCR: Accuracy and precision issues in complex samples.

Digital PCR (dPCR)

• Overview:
  • Invented before qPCR but previously unaffordable and technically challenging.
  • Direct molecule counting method for quantifying DNA and RNA.
  • Example: Duplex reaction with Staph aureus and human genomic DNA.
  • Error Bars: Represent 95% confidence intervals.

dPCR Technique

• Partitioning:
  • Standard 20 µL qPCR reaction is divided into thousands of sub-reactions (droplets).
  • Each droplet undergoes amplification in a thermal cycler.
  • Analyze presence/absence of target.

Data Analysis

• Graphical Representation:
  • Plot fluorescence amplitude vs. droplet number.
  • Identify positive (target present) and negative (target absent) droplets.
  • Threshold set for defining positivity (e.g., 1000 fluorescent units).

Counting Molecules

• Example of Samples:
  • Sample 1: 0 positive droplets (negative for target).
  • Sample 2: 6 positive droplets (estimated 6 targets).
  • Sample 3: 34 positive droplets (estimated 34 targets).
  • Sample 4: 70 positive droplets (estimated 70 targets).
• Poisson Correction:
  • Important for accurate counting due to likelihood of multiple molecules in one droplet.
  • Formula: Concentration (copies/µL) = -ln(negative droplets / total droplets) / volume of droplets.

Sample Corrections

• Sample 2: Corrected to 6.2 copies.
• Sample 3: Corrected to 38 copies.
• Sample 4: Corrected to 96 copies.
• Important Note: Analyze at least 10,000 droplets for better accuracy.

Final Calculation Example

• Sample with 6898 positive droplets and 13232 negative droplets:
  • Using Poisson equation: 494 copies/µL, leading to 9880 total copies for the reaction.
• Software: Available to automate calculations.

Advantages of dPCR

• Precision and accuracy.
• Absolute quantification without standards.
• High-order multiplexing capability.
• High sensitivity in complex samples.
• Robustness compared to other methods like qPCR.

Conclusion

• For more information:
  • Download Bulletin 6407 from BioRad.com.
  • Access additional resources from Springer Publishing.
• Thank You!
  • Invitation to explore further modules and applications.