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Klinik für Innere Medizin IHämatologie, Onkologie und Stammzelltransplantation

Digital PCR

dPCR
Fluorescent ddPCR droplets

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 system from Bio-Rad and a recently acquired Stilla Naica Crystal Digital PCR System 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 reactions (approx. 1 nL volume for the QX100; slightly lower for the Stilla). 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 up to 30,000 aqueous droplets within an oil carrier. 

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

On The Bio-Rad system, the sample is placed on the Droplet Reader where the droplets are examined individually, providing an independent digital measurement. Depending on their signal, the droplets are scored as either positive or negative for the marker. Based on the number of positive droplets and the number of partitions/droplets overall, the number of target molecules which were originally present, in the reaction. can be calculated ith the help of the Poisson distribution.

 


Stilla Naica System - Digital PCR

cdPCR computed FAM pos and neg droplets (cutout)

In addition to its Bio-Rad QX100 Droplet Digital PCR system Lighthouse also has a Stilla Naica Crystal Digital PCR system.

Digital PCR (cdPCR as well as ddPCR) is related to quantitative PCR (qPCR), 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%)

There are some specific differences between the two systems. 

On the Stilla system the sample first flows through a network of microchannels and is partitioned into a large 2D array of up to 30,000 individual droplets, also called a droplet crystal. PCR is then performed on-chip and the droplets are scanned to reveal those which contain amplified targets. The last step consists of counting the number of these positive droplets to precisely extract the absolute quantity of nucleic acids.

cdPCR crystal droples with FAM neg and pos droplets

On the Stilla system the sample first flows through a network of microchannels and is partioned into a large 2D array of up to 30,000 individual droplets, also called droplet crystal. PCR is the performed on-chip and the droplets are scanned to reveal those which contain amplified targets. The last step consists of counting the number of these droplets to prececiesely extract the absolute quantity of nucleic acids. 

 

3D plot of FAM, VIC and Cy5 negatives, single, double and triple positives

Workflow

  1. Sample pipetting: Only one pipetting step is required to load the reaction mixes into the wells of the Sapphire (or Opal) Chips.
  2. Droplet crystal generation and amplification: Once the chips are placed into the Naica Geode, launch the combined partitioning and thermocycling program. Crystals of up to 30,000 droplets are created from each sample, PCR amplification is performed immediately after crystal generation.
  3. Crystal reading: Transfer the chips to the Naica Prism3 instrument. Crystals are read using 3 fluorescence channels.
  4. Data analysis: The crystal reader program Crystal Miner software automatically measures the concentrations of targeted nucleic acids. Analyze your data and explore your crystals using the program.

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 6628

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

Bio-Rad 6407

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. 


General Applications Guide

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

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