Digital Droplet PCR

The Genome Analysis and Technology core has a Bio-Rad QX200™ Droplet Digital™ PCR System which either researchers or the core  operate. This system divides a sample into 20,000 droplets which contain no nucleic acid or one or more molecules of nucleic acid. After performing typically 40 cycles of PCR, each droplet is read to see if it contains nucleic acid. By counting the number of droplets with and without detectable nucleic acid, the number of positive droplets is fitted to a Poisson distribution to calculate the original concentration of nucleic acid.

Droplets are nanoliter size. Droplets from multiple wells of a sample can be combined for higher sensitivity. 96 samples can be run in 5 hours. Suggested sample size 25 µL. Use TaqMan hydrolysis probes or EvaGreen dye.

Because single molecules of nucleic acid give a signal, digital PCR has high sensitivity. The counting method for estimating the number of nucleic acids gives high precision readings. These two characteristics make digital PCR an excellent technique for a number of applications:

  • quantitation of DNA and RNA without standard curves over a range of 1 to 120,000 copies (400 ng of human DNA) /20 µL
  • detect mutations as low as 1 in 100, 000
  • compare expression levels of a gene between samples
  • copy number variation (CNV); 1.2 fold variations detectable
  • miRNA detection and possible use of miRNA as biomarkers
  • pathogen detection
  • improved Next Generation sequencing data by improving sample quantitation
  • possible monitoring of cancer therapies

Current uses at the University of Virginia include:

  • quantitation  of libraries for Nex Gen sequencing
  • characterization of microDNA (Dr. Dutta)
  • gene level measurement (Drs. J-P Liu and Zeitlin)
  • detection of rare gene rearrangments (Dr. Y-H Wang)