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Klinik für Innere Medizin IHämatologie, Onkologie und Stammzelltransplantation

Digital PCR

dPCR

 

Digital PCR is a form of quantitative PCR based on end-point measurements. It has distinct advantages over QPCR for specific types of assays:

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

Lighthouse has both a QX100 Droplet Digital PCR (ddPCR) system from Bio-Rad and a recently acquired Stilla Naica Crystal Digital PCR System (cdPCR) available for you to use. Specific differences between the two systems can be found below.

 

What is Digital PCR?

Digital PCR excels in the measurement of low copy numbers, and has a linear range of 5 logs (from 1 to  approximately 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, dPCR allows rare molecules to be detected within complex mixtures much more efficiently than standard QPCR. This is particularly useful in such applications as detecting circulating tumor DNA in cancer patients, where there are overall very few molecules of DNA to detect, within a large background of wild type DNA.

 

Digital PCR: Lighthouse System Comparisons

System and Detection Method Fluorophores Hydrolysis (Taqman) Probes Intercalating Dyes (EvaGreen)

Additional Reaction Components Needed

Examples of Compatible Mixes

Bio-Rad QX100

(Probes)

 FAM

 HEX / VIC

yes not available - ddPCR Supermix for Probes (no dUTP)

Stilla Naica

(Probes)

 FAM

 HEX / VIC

 CY5

yes -

Fluorescein
(Reference dye for detection of negative droplets)

 

Probes mixes:

PerfeCTa Multiplex Tough Mix

qScript XLT 1-Step RT-PCR Kit

Stilla Naica

(EvaGreen)

EvaGreen - yes

EvaGreen dye (20x in water) 
(Intercalating dye for DNA detection)

Alexa Fluor 647 Dextran
(Reference dye for detection of negative droplets)

EvaGreen mix:

PerfeCTa qPCR ToughMix UNG

 

Digital PCR: How does it work?

Digital PCR works by partitioning the QPCR reaction into very many small reaction volumes (approx. 1 nL volume for the QX100). Lighthouse systems create many small droplets within a water-in-oil emulsion, located in either a cartridge well (BioRad QX100) or on a special optical slide (Stilla Naica).

ddPCR droplets in a tube

 

Droplets are generated as 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.

 

 

Further Information

Some useful documents about dPCR:

Stilla Naica Crystal Miner User Manual v.2.0 User manual for the Naica Crystal Miner software
Stilla Naica Crystal Reader User Manual 2.0 User manual for the Naica Crystal Reader

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 Lighthouse Core Facility. 

Download General Applications Guide

General Applications Guide for droplet digital PCR with many application descriptions and tips and tricks

Download

 

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:

Email:

Online Booking Calendar (new!)
Facslab-email

 

Online Reservations (for machines others than cell sorters until July. 31st. 2020):

Booking Calendar (intranet only)
 

Head of Facility:

Marie Follo, Ph.D.
Telephone: 0761 270-77697
Telefax: 0761 270-63780
Email: Marie Follo

 

Address:

Universitätsklinikum Freiburg
Lighthouse Core Facility

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

www.core-facility.de