Chapter 7: Applications of ddPCR in Genomic Analysis

Digital PCR (ddPCR) enables absolute quantification of nucleic acids without the need for calibration curves.
This makes it a powerful tool in genomic applications where precision is critical, such as:

  • Copy Number Variation (CNV) detection
  • Rare allele or mutation detection

Copy Number Variation (CNV) Detection

Copy Number Variation (CNV) refers to a gain or loss of gene copies in the genome. ddPCR quantifies the number of gene copies in a sample and compares it to a reference gene with known normal copy number (usually 2).

The relative copy number is calculated as:

\[ \text{Copy Ratio} = \frac{\text{Target Gene Concentration}}{\text{Reference Gene Concentration}} \]

Example:

target <- 10.1 # estimated concentration of target gene (copies/μL)
reference <- 5.0 # estimated concentration of reference gene (copies/μL)
copy_ratio <- target / reference
round(copy_ratio, 2)
## [1] 2.02

Interpretation

Copy Ratio Interpretation
~1.0 Normal diploid copy
~0.5 Heterozygous deletion
~2.0 Gene duplication

ddPCR is especially useful for detecting subtle CNVs that may not be distinguishable by qPCR or arrays.

Rare Allele Detection

ddPCR can detect low-frequency mutations, even at < 0.1% mutant allele frequency (MAF). This is crucial for applications such as:

  • Detecting resistance mutations in cancer (e.g., EGFR T790M)
  • Tracking minimal residual disease (MRD)
  • Analyzing circulating tumor DNA (ctDNA)

Dual-probe strategy

Each droplet is tested for both:

  • Wild-type (WT) using one probe (e.g., HEX)
  • Mutant (MT) using another probe (e.g., FAM)
  • The Mutant Allele Frequency (MAF) is calculated as:

\[ \text{MAF} = \frac{\text{Mutanat Copies}}{\text{Mutant Copies + Wild-Type Copies}} \]

Example:

mutant <- 6
wt <- 994

maf <- mutant / (mutant + wt)
round(maf * 100, 3)
## [1] 0.6

This demonstrates ddPCR’s ability to detect rare mutations in a background of wild-type DNA.

Summary

Application Measurement ddPCR Advantage
CNV Detection Copy ratio (Target / Reference) Absolute quantification without a standard curve
Rare Allele Detection Mutant Allele Frequency (MAF) Sensitive detection of <0.1% MAF