Digital PCR: the most accurate PCR method for assessing the genetic integrity of Stem Cells

Digital PCR is the most accurate PCR method for assessing the genetic integrity of stem cells through the detection of Copy Number Variants (CNVs).

At Stem Genomics, we help you assess the genetic integrity of your stem cell lines with digital PCR-based tests designed to suit your organization and the specific features of your cell lines.

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Digital Droplet PCR technology

Digital PCR - A key tool for genomic integrity testing

Digital PCR (dPCR) is the most accurate PCR method for assessing the genetic integrity of stem cells through the detection of Copy Number Variants (CNVs).

Why?

Using specific primer sets and fluorescent probes, dPCR amplifies targeted genomic fragments in thousands of individual reactions, thus allowing nucleic acid quantification and detection with high precision.

The power of dPCR relies on the random distribution of the sample and assay mixture into a very large number of distinct sub-reactions. Such partition allows multiple simultaneous endpoint PCR amplifications through the measurement and counting of positive (i.e. containing the amplified target) and negative (i.e. without the amplified target) partitions. The term ‘digital’ defines this binary result counting method. Positive reactions, which contain at least one copy of the target DNA fragment, are identified by their fluorescence signal. Comparison of the number of positive and negative reactions allows the target quantification using the Poisson distribution method. Then, the absolute number of molecules present in the sample is calculated by comparison with the results obtained for a reference gene.

These features make of dPCR an ideal tool for CNV detection with high sensitivity and precision.
 

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qPCR versus dPCR

Comparison with quantitative PCR

Although it uses the same amplification reagents as dPCR, quantitative PCR (qPCR) is based on the analysis of a single homogeneous reaction mixture. Therefore, the number of template DNA copies is measured by real-time monitoring of the amplification at the exponential phase of the reaction, providing only a relative CNV quantification.

Main advantages of dPCR

The main advantages of dPCR compared with conventional qPCR methods are:
• Absolute quantification of nucleic acids without relying on external references (e.g. standard calibration curves);
• Robustness to variations (thus, dramatic reduction of inter- and intra-assay variability) and tolerance to PCR inhibitors;
• High precision in CNV detection thanks to reaction partitioning (i.e. small fold differences can be revealed and this can be interesting for the detection of low-frequency mosaic abnormalities).

 

https://www.stemgenomics.com/detect-abnormalities-in-hpscs-0
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