ddPCR                ddPCR

HOW does it work? (Technology)

  • PCR reaction containing fluorescently labeled primers (FAM, and HEX or VIC) or EvaGreen (SYBR-like dye) is partitioned into 20,000 droplets.
  • End point PCR amplifies the DNA within each droplet.
  • Droplets are separated and read individually.
  • Droplets with labeled target molecules read as positive, while droplets without target are negative.
  • Poisson statistical algorithm used to adjust for droplets with two or more targets for count. 

    

WHEN would ddPCR be used instead of qPCR? (Advantages)

  • When you need absolute quantitation versus indirect quantitation - No standard curve needed
  • To detect rare events
  • To detect fine variations such as copy numbers
  • To detect smaller fold change differences
  • Increased precision with more replicates
  • Less sensitive to PCR efficiency

WHAT do you use it for? (Applications)

  • Copy Number Variation
  • Rare Mutation Detection
  • Absolute Quantification Nucleic Acids
  • Identify Under-recognized Somatic Mosaicisms
  • Single-Cell Gene expression analysis
  • Environmental Samples, monitor invasive species, detect viruses and microbes
  • Detection tumor cfDNA in blood
  • Illumina library generation: maximize rarer expressing genes by PCR of droplets before going on to prep.

WHO do you contact in CGRB? 

 Anne-Marie Girard and Jessica Nixon