The human genome is of extraordinary complexity, with its three billions of
DNA base pairs that form the thousands ofgenes that make up our biology.
Understanding this complexity requires powerful tools.
Digital PCR is a powerful new tool forquantitative DNA measurements.
Like real-time PCR, digital PCR uses a simple sample preparation that utilizes
TaqMan primers and probes and an optimized supermix.
Real-time PCR provides relative quantification.
In contrast, digital PCR provides absolute quantification of target DNA
sequences in the sample.
Let's see how Droplet Digital™ PCR achieves precise DNA measurements.
The key is sample partitioning. With Droplet Digital PCR, reaction mixes are
partitioned into approximately 20,000 evenly-sized droplets, each 1 nl
in volume.
PCR is performed on each individual droplet.
If a droplet contains the target sequence, it will be amplified and a TaqMan
reporter dye will emit a fluorescent signal.
If the target sequence is not present in a droplet, no PCR will take place and
only weak residual fluorescence is emitted.
By scoring each individual droplet for a fluorescent signal, Droplet Digital PCR
achieves absolute quantification with a high level of precision and
single-molecule sensitivity.
To generate digital results, samples are amplified to endpoints in a conventional
thermal cycler. Droplets that contain the target DNA sequence are read as
positive.
Those that do not are read as negative. The fraction of positive droplets
determines the concentration of the target DNA sequence in the sample. Now
let's look at how the Bio-Rad® QX100™ Droplet Digital™ PCR system
performs digital PCR with unrivaled precision and sensitivity.
The QX100 system uses a simple workflow that requires minimum hands-on
time and procedures that are easy to perform.
First, 20 µl each of eight prepared samples are transferred to the
droplet generation cartridge.
Droplet generation oil is then loaded into the corresponding channels of the
cartridge. There are eight oil channels, one for each of the samples.
Once the cartridge is loaded with prepared samples and oil, it is placed in the QX100
droplet generator.
Using microfluidic technology, the droplet generator combines the samples and
oil to create an emulsion of approximately 20,000 monosized
droplets for each sample.
Every droplet contains a nanoliter volume of the sample, including any
target sequences that may be present.
After the droplets are generated, the emulsified samples are transferred to a
standard 96-well PCR plate using a standard 8-channel pipet. The plate
is then loaded onto a thermal cycler where PCR is performed to amplify the
target DNA to endpoint.
When PCR is complete, the plate is loaded onto the QX100 droplet
reader.
The QX100 droplet reader is an automated liquid handling device
equipped with optimized microfluidics designed for precise, reproducible
detection of target DNA sequences,
droplet by droplet. The droplet reader automatically sips each sample, singulates
all the droplets from a sample, and then streams them in a single file past a
two-color fluorescence detector. The detector reads each droplet in turn at the rate of
1,000 droplets per second
and determines which droplets emit fluorescence and which do not.
As each droplet is read, the data are recorded graphically by Quantasoft™ software. The
software records the positive and negative droplets in each sample and
plots the droplet-by-droplet fluorescence. This allows the data to be
analyzed and visualized according to specific application and research
requirements.
The accuracy, sensitivity, and precision of Droplet Digital PCR enable a wide
variety of applications that require accurate, absolute quantification.
Examples of these applications include copy number variation,
mutation detection,
and gene expression.
QX100 Droplet Digital PCR —
the future of quantitative PCR is here, and it's digital.
Be part of it.
For more information about the QX100 Droplet Digital PCR system
visit our website, or contact us directly by phone or email. 71