Droplet Digital PCR

Report
Droplet digital PCR
Overview and applications
Genetic Technologist Training Day
Thursday 20th November 2014
Natalie Brace
Contents
• Introduction into Digital PCR
• Workflow
• Advantages of ddPCR
• JAK2 testing
• Future developments
What is droplet digital PCR?
• A real-time PCR reaction that is partitioned into many droplets – a PCR
reaction happens in each droplet.
• A portion of these reactions contain the target molecule while others do
not.
• Positive reactions are counted.
• Fraction of negative reactions is used
to deduce the absolute number of
target molecules in the sample.
•
Currently in use to test for JAK2
V617F mutation.
ddPCR work flow
1) QX100
Droplet generator
2) Bio-Rad
Thermal cycler
3) QX100
Droplet reader
ddPCR work flow
1. Prepare sample and reagent mixture
• Primers and fluorescent probes are combined with the Biorad ddPCR
supermix
• DNA is then added to create PCR ready samples
• Loaded into individual wells of the 8 channel disposable droplets
droplet generator cartridge
Sample
oil
2. Make droplets
• Place loaded cartridge onto QX100 droplet generator
• Partitions reagents and sample to create up to 20,000 droplets per
well
Oil
Sample
Oil
ddPCR work flow
3. PCR droplets on Thermal Cycler
• Transfer the droplets from the cartridge to a 96 well PCR plate
• Seal the plate using Biorad PX1 plate sealer
• Perform PCR to endpoint (40 cycles) using a thermal cycler
4. Read droplets
• Post PCR, load 96 well plate onto the QX100 droplet reader
• Droplets are streamed single file past a two colour optical detection
system to determine if they contain a target (positive) or not (negative)
• Analyse concentrations using QuantaSoft software
Key advantages of ddPCR
• Absolute quantification means no standard curve is needed.
• Cost effective when compared with quantitative PCR.
• Highly flexibility technique - High throughput with single well measurements for 96
samples
 - High sensitivity, dependent on the number of droplets
Droplet Digital PCR platforms
20K droplets/20ul PCR reaction
0.9nL droplets
Biorad ddPCR Supermix
(~£3 per reaction)
5-10M droplets/25ul PCR reaction
0.5pL droplets
Open system
(~£19 per reaction)
JAK2- Janus kinase 2
•
Protein involved in JAK/STAT signalling pathway.
•
The JAK2 protein is especially important for controlling the production of blood
cells from hematopoietic stem cells.
•
The V617F JAK2 gene mutation results in the production of a JAK2 protein that is
constantly activated, this leads to the overproduction of abnormal blood cells.
•
Excess platelets can cause abnormal blood clotting.
•
With so many extra cells in the bloodstream, abnormal blood clots are more likely
to form. In addition, the thicker blood flows more slowly throughout the body,
which prevents organs from receiving enough oxygen.
•
Somatic JAK2 V617F mutation is found several chronic myeloproliferative
neoplasms
• – Polycythemia vera (97%), essential thrombocythemia (57%), and chronic
idiopathic myelofibrosis (50%).
Previous JAK2 testing strategy
Two tier strategy;
Diagnostic Allele Specific Oligonucleotide (ASO) PCR (~£3)
• TAT- 14 days.
• Lots of re-runs due to issues with ABI- size standard drop outs etc.
• ‘Equivocal’ results sometimes given by ASO (Ratios between 1.5% and
5.0%).
Follow up RQ-PCR(~£30)
•
Resolve ‘equivocal’ results.
•
Monitor Post transplant patients .
•
Monitoring for positive patients.
•
Sensitivity to detect JAK2 V617F down to 0.09%.
•
Relatively few patients so batching required; turn around times may be
up to 2 months.
ddPCR JAK2 testing strategy and
service
• Use a single test to replace the two tier strategy with the
aim to
 Improve turnaround times
 Improve sensitivity
• To reliably detect 1% mutation load using 1 well of QX100
 1% is the clinically relevant mutation load according to Best
Practice Guidelines (Bench et al.2013)
• Approx. 200 samples a month received into the laboratory
• 3 runs of 20 patients each week usually sufficient to clear all
samples and repeats for positives
• Regular runs means fast confirmation of positives.
JAK2 V617F ddPCR assay using
QX100
We use an assay designed by Quantalife-
This is a Duplex assay using hydrolysis probes specific for JAK2
wild type (VIC) and JAK2 V617F mutation (FAM)
JAK2 Results
•
QX100 generates a maximum of 20,000 droplets
•
Its possible that when droplets are generated some
droplets contain more than one target DNA.
•
Positive and negative reactions are counted by the
QX100 reader and are fit to a Poisson distribution to
account for the possibility of having more than one
target in each droplet.
Future ddPCR uses and
developments
• KIT mutations in Myeloproliferative Disorder (MPD)
patients – SOP being developed
• BCR-ABL testing for Chronic Myeloid Leukemia
(CML) using the Raindance platform – higher
sensitivity – ongoing research
• Fetal sexing using the QX100- new project
Thank you for
listening.
Any Questions?

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