Mentype® DigitalQuant

Sensitive quantification for advanced DNA ratio analysis

Features

Absolute quantification with no need of technical replicates
Quantification of target DNA ratio ≤ 0.1 %
Performed on standard dPCR systems
Application e. g. for chimerism, graft rejection, or cell product research

Following the initial genotyping, Mentype^® DigitalQuant is employed for precise quantification of DNA ratios, such as in chimerism or transplantation studies. This approach uses digital PCR technology and offers higher sensitivity and reproducibility through optimized DNA input.

Biomarkers

Insertion-deletion polymorphism (DIPs or INDELs), SRY, Reference gene

Product Specifications

Panel
59 allele specific duplex assay

Reactions
3 allele specific assays per sample (3 reactions)

Internal controls
Reference gene or SRY

PCR controls
2 (PC extra available, NTC)

Sample input
50 ng gDNA from peripheral blood or bone marrow

Sensitivity
0.1 %

Turnaround time
~ 2.5 h after nucleic acid preparation

Detection
Absolute qualitative

To be used with
Bio-Rad QX100/200, Applied Biosystems QuantStudio^TM 3D/ AbsoluteQ^TM

Scientific Background

The Mentype^® DigitalQuant approach employs the highly sensitive digital PCR technology that allows for absolute quantification of DNA samples. Specific for digital PCR (dPCR) is the sample-partitioning into a plethora of nano droplets or cavities. Each separate compartment contains nano liters of the sample of interest and during thermal cycling it functions as a separate PCR amplification chamber. Depending on how many copies of the target DNA molecules have been divided into the compartments (zero, one or more copies), a multitude of replicates is generated in each PCR run. Using Poisson statistics, the absolute number of starting copies can be determined very accurately. After thermal cycling, each compartment is automatically analyzed and determined as positive or negative fraction. Because the digital PCR uses end-point detection of the amplification product, calibration curves are not necessary.

Mentype^® DigitalQuant assays were developed to quantitatively analyze identified DIP loci (deletion insertion polymorphisms, INDEL). These assays represent a set of 29 different DIP alleles and are provided as duplex mixes either in combination with ß-globin as an active reference (REF) or with SRY as a Y-chromosome specific marker. Additionally, a combination of SRY/REF is provided as an assay.

Product References

Picard, C. et al. New methods for the quantification of mixed chimerism in transplantation. Front. Immunol. 14, https://doi.org/10.3389/fimmu.2023.1023116 (2023)
Fortschegger, M. et al. Detection and Monitoring of Lineage-Specific Chimerism by Digital Droplet PCR-Based Testing of Deletion/Insertion Polymorphisms. Biol Blood Marrow Transplant 26, 1218-1224, https://doi.org/10.1016/j.bbmt.2020.02.016 (2020)
Mika, T. et al. Digital droplet PCR-based chimerism analysis for monitoring of hematopoietic engraftment after allogeneic stem cell transplantation. Int J Lab Hematol. 41, 615-621. https://doi.org/10.1111/ijlh.13073 (2019)
Pedini, P. et al. Droplet Digital PCR: A New Technology for Detection and Quantification of Chimerism After Allogenic Hematopoietic Stem Cell Transplantation. Biomed J Sci & Tech Res. DOI: 10.26717/ BJSTR.2019.13.002420. (2019)
Stahl, T. et al. Digital PCR Panel for Sensitive Hematopoietic Chimerism Quantification after Allogeneic Stem Cell Transplantation. Int. J. Mol. Sci. 17. https://doi.org/10.3390/ijms17091515 (2016)
Stahl, T. et al. Digital PCR to assess hematopoietic chimerism after allogeneic stem cell transplantation. Experimental Hematology 43, 462-468. https://doi.org/10.1016/j.exphem.2015.02.006 (2015)