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Klinik für Innere Medizin IHämatologie, Onkologie und Stammzelltransplantation

Droplet Digital PCR

ddPCR

Lighthouse has a QX100 Droplet Digital PCR (ddPCR) system from Bio-Rad, which can be used in combination with TaqMan / Hydrolysis Probe-based assays. Currently, up to two colors can be used simultaneously (FAM and VIC/Hex)

Digital PCR is related to quantitative PCR, but has distinct advantages for certain types of assays:

  • Absolute quantification
  • Detection of rare target sequences
  • Single cell analysis
  • Detection of small fold changes (+/-10%)

ddPCR excels in the measurement of low copy numbers, and has a linear range of 5 logs (from 1 to 100,000 copies per reaction). The linearity of the process is what allows the detection of small fold changes.

Because of its ability to absolutely quantify the number of molecules present within a sample, the use of a reference gene is not an absolute requirement in digital PCR. In addition, ddPCR allows rare molecules to be detected within complex mixtures much more efficiently than standard QPCR. This is particularly useful in such applications as looking at the levels of minimal residual disease in cancer patients, where there is a large background of negative cells.

 

Digital PCR: How does it work?

Digital PCR works by partitioning the QPCR reaction into very many small reactions (approx. 1 nL volume for the QX100), through either a physical separation as used in chip-based systems, or through the creation of many small droplets in an emulsion as is the case with ddPCR. 

ddPCR droplets in a tube

The Droplet Generator forms an emulsion in which the sample is partitioned into 20,000 aqueous droplets within an oil carrier. The droplets have an average volume of 1 nanoliter each.

 

 

ddPCR droplets with green fluorescence in a tube

The target and background DNA are randomly distributed throughout the droplets as the droplets are formed. The sample can then be run on an ordinary thermal cycler.

 

 

Droplets with or without green fluorescence

After the PCR amplification step, each droplet provides a fluorescent positive or negative signal indicating whether the target was present or not within the droplet after partitioning.

 

 

Reader's signal of green and non green droplets

The sample is placed on the Droplet Reader where the droplets are examined sequentially, similar to a flow cytometer, providing an independent digital measurement. The droplets are scored as either positive or negative for the marker.

The fraction of positive droplets can be used with the Poisson Distribution to calculate the number of target molecules which were originally present in the reaction. The design of hydrolysis probes and primers is critical to the success of the experiment. The Core Facility has a copy of the Beacon Designer program to help with primer and probe design and seeing that these parameters are met.

Some Useful Documents about ddPCR:

Bio-Rad 6260

Bio-Rad technical note on detecting rare mutant alleles with ddPCR

RED application guide

Bio-Rad application guide on using ddPCR for Rare Event Detection

Bio-Rad 6277

Bio-Rad technical note on looking at copy number variation by ddPCR

For more information about using the machine or for help in designing possible probes/primers, please contact the Core Facility. 

Further Informations

 

Universitätsklinikum Freiburg
Zentrum für Translationale Zellforschung (ZTZ)
Department of Medicine I
Tumorzentrum Freiburg - CCCF/DKTK
Center for Chronic Immune Deficiency - CCI

Contact | Information

 

FACS-Sorting Booking:

Telephone: 
Arias: 0761 270-63770
MoFlo: 0761 270-77680

Email:
facslab@uniklinik-freiburg.de

 

Online Reservations (for machines others than cell sorters):

Booking Calender (intranet only)
MoFlo Calender (read only)

 

Head of Facility:

Marie Follo, Ph.D.
Telephone: 0761 270-77697
Telefax: 0761 270-63780
marie.follo@uniklinik-freiburg.de

 

Address:

Universitätsklinikum Freiburg
Lighthouse Core Facility

Zentrum für Translationale Zellforschung (ZTZ)
Breisacher Straße 115
D-79106 Freiburg

www.core-facility.de