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Digital Droplet PCR - How does it work?04:04

Digital Droplet PCR - How does it work?

Digital Droplet Polymerase Chain Reaction (ddPCR ) is a new generation PCR method which allows for tens of thousands of PCR reactions from one sample occur simultaneously.



How it worksEdit

DdPCR uses the water-oil emulsion principle to form around 20,000 droplets (1 nanoliter), each of which carries out a given number of PCR reactions. Here is a step-by step summary of how ddPCR works:

1. DNA sample is divided into ~20,000 reaction droplets.

2. Each droplet contains specific primers, added to bind to target DNA sequence and then serve as binding sites for TaqMan (specifically Taq Polymerase). A TaqMan reporter dye emits a fluorescent dye as it aplifies the DNA sequence. No flurorescent dye is released if there is no target sequence present in a droplet. The TaqMan probe is composed of a non-specific sequence complementary to the DNA in the sample along with two molecules: a quencher and a fluorophore. The fluorophore does not emmit any fluorescence unless it has been detached from the TaqMan probe, making the amount of total fluorescence emitted in the sample directly proportional to the amount of amplification (see Slide Figure 3). 

3. DdPCR will then quantify fluorescence from each droplet, whcih allows it to achieve absolute quantification with single molecule sensitivity.

See video 1 for a visual and mroe detailed summary of the steps above.

Applications in GenomicsEdit

Due to its ability to make absolute measurements of targeted DNA sequences in the sample, ddPCR has wide-applications in the field of Genomics (Slide 1):

  • Visualization of ddPCR results, intended for CNV quantification
  • 2D plot of mutant cell lines' DNA. DdPCR enables one to detect rare mutations (upper right corner)
  • TaqMan probe chemistryGo to Source: Wikipedia

1. Quantification of Copy Number Variation (CNVs)

- Duplications or deletions of DNA segments [1]

2. Identification of Rare mutations [2]





ReferencesEdit

1. Boettger et al., "Structural haplotypes and recent evolution of the human 17q21.31 region", Nature Genetics 44, 881–885 (2012)

2. Chen et al., Molecular Therapy Nucleic Acids (2013) 2, e109

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