HOW does it work? (Technology)
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PCR reaction containing fluorescently labeled primers (FAM, and HEX or VIC) or EvaGreen (SYBR-like dye) is partitioned into 20,000 droplets.
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End point PCR amplifies the DNA within each droplet.
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Droplets are separated and read individually.
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Droplets with labeled target molecules read as positive, while droplets without target are negative.
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Poisson statistical algorithm used to adjust for droplets with two or more targets for count.
WHEN would ddPCR be used instead of qPCR? (Advantages)
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When you need absolute quantitation versus indirect quantitation - No standard curve needed
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To detect rare events
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To detect fine variations such as copy numbers
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To detect smaller fold change differences
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Increased precision with more replicates
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Less sensitive to PCR efficiency
WHAT do you use it for? (Applications)
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Copy Number Variation
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Rare Mutation Detection
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Absolute Quantification Nucleic Acids
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Identify Under-recognized Somatic Mosaicisms
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Single-Cell Gene expression analysis
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Environmental Samples, monitor invasive species, detect viruses and microbes
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Detection tumor cfDNA in blood
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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 or Katie Carter