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Digital Droplet PCR (ddPCR） (CAT#: STEM-MB-0188-WXH)

Introduction

Digital droplet PCR or ddPCR is a method that provides ultrasensitive and absolute nucleotide concentration, unlike qPCR where results can vary across replicates. ddPCR can be used to quantify DNA sequences that are rare, for example, rare alleles or mutations. ddPCR also does not require a reference or standard curve, which can be time-consuming.
ddPCR has the following benefits for nucleic acid quantification:
Unparalleled precision
Increased signal-to-noise
Removal of PCR eficiency bias
Simplified quantification - a standard curve is not required for absolute quantification

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Principle Applications Procedure

Principle

ddPCR uses a water-oil emulsion droplet technology that fractions the PCR reaction sample into approximately 20,000 droplets. Each droplet contains the material required for PCR amplification. Following the PCR reactions each droplet is analyzed by a droplet reader, which measures the fluorescence amplitude of each droplet. The fraction of fluorescent PCR-positive droplets is determined and then analyzed using Poisson statistics to determine the concentration of the original template DNA in the sample.

Applications

• Liquid biopsies: noninvasive tests that are able to detect cancer cells (circulating tumor cells, CTCs) or DNA shed from tumors (circulating tumor DNA, ctDNA) in the blood.
• Copy Number Variation: Provide accurate CNV quantification, studies profiling CNV variations in populations of interest.
• Rare Sequence Detection:
Detection of cancer below the level detectable by current tests.
Monitoring for new mutations and duplications in cancer as they arise.
Detection of viral loads.
• Gene Expression.
• Single-cell analysis.
• Next-Generation Sequencing (NGS).

Procedure

1.Droplet generation
2.PCR amplification
3.Droplet reading
4.ddPCR data analysis

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