technology feature
`
`digital Pcr on chips
`digital Pcr in droplets
`thinking digitally
`
`542
`543
`544
`
`Digital PCR hits its stride
`Monya Baker
`
`As the less familiar cousin of quantitative PCR moves mainstream, researchers have more options to choose from.
`
`A few years ago, Ramesh Ramakrishnan
`had to spend so much time explain-
`ing what digital PCR was that he had to
`rush through his explanations of appli-
`cations when he gave talks at meetings.
`Now, he says, most audiences are at least
`familiar with the term, even if they have
`not performed the technique them-
`selves. “It’s no longer an exotic thing,”
`says Ramakrishnan, director of R&D at
`Fluidigm Corporation.
`The strategy for digital PCR (dPCR)
`has been summarized as ‘divide and con-
`quer’: a sample is diluted and partitioned
`into hundreds or even millions of sepa-
`rate reaction chambers so that each con-
`tains one or no copies of the sequence of
`interest. By counting the number of ‘posi-
`tive’ partitions (in which the sequence is
`detected) versus ‘negative’ partitions (in
`which it is not), scientists can determine
`exactly how many copies of a DNA mol-
`ecule were in the original sample. Among
`other applications, researchers have
`used digital PCR to distinguish differen-
`tial expression of alleles1, to track which
`viruses infect individual bacterial cells2,
`to quantify cancer genes in patient speci-
`mens3 and to detect fetal DNA in circulat-
`ing blood4.
`The concept behind digital PCR was
`first described in 1992 (ref. 5). A few years
`later, Bert Vogelstein and Ken Kinzler
`at Johns Hopkins University named the
`technique and showed that it could be
`used to quantify disease-associated muta-
`tions in stool from patients with colorec-
`tal cancer. But although the theory was
`simple, its implementation was not. Initial
`demonstrations were performed in com-
`mercially available 384-well plates with
`5 microliters per partition, requiring vol-
`umes of reagents that would daunt most
`researchers6.
`
`Advances in nanofabrication and micro-
`fluidics have now led to systems that pro-
`duce hundreds to millions of nanoliter- or
`even picoliter-scale partitions. Academic
`technology developers have described
`several implementations, but so far only
`a handful of companies have commercial-
`ized products or announced plans to do so
`(Table 1). Fluidigm and Life Technologies
`create reaction chambers within specially
`designed chips or plates. Bio-Rad and
`RainDance sequester reagents into indi-
`vidual droplets.
`
`higher costs, higher precision
`The most popular PCR technique to mea-
`sure the presence and concentration of a
`DNA sequence is not digital PCR but its
`more familiar cousin, real-time quantita-
`tive PCR (qRT-PCR, or qPCR). In qPCR,
`DNA is copied until it produces a certain
`level of signal; the number of amplification
`cycles needed to reach this point is then
`used to calculate how many DNA mol-
`ecules with the particular sequence were
`originally present relative to other DNA
`molecules in the sample.
`Digital PCR uses the same primers and
`probes as qPCR but is capable of higher
`s e n s i t i v i t y a n d
`precision. In stan-
`dard implementa-
`tions, qPCR cannot
`dist inguish gene
`expression differ-
`ences or copy num-
`ber variants smaller
`than about twofold.
`Identifying alleles
`with frequencies of
`less than about 1%
`is difficult because
`such tests would also
`detect highly abun-
`
`dant common alleles with similar sequenc-
`es. In contrast, dPCR can measure a 30%
`or smaller difference in gene expression,
`distinguish whether a variant occurs in
`five versus six copies and identify alleles
`occurring at a frequency of one in thou-
`sands. It can also be used to standardize
`qPCR assays.
`The more partitions, the greater the
`resolution. “If you want to distinguish
`between 2 and 3 copies, you need 200
`chambers. If you want to distinguish
`between 10 and 11, you need 8,000,”
`explains Mikael Kubista, CEO of TATAA
`Biocenter, which provides services and
`training in both qPCR and digital PCR. In
`principle, one could also get similar preci-
`sion by doing 8,000 replicates of qPCR, he
`says, but such experiments are impractical.
`Jim Huggett is the science leader for
`nucleic acids metrology at LGC, a labo-
`ratory services and measurement stan-
`dards organization in the UK. His team
`has directly compared the two techniques
`across several DNA templates and other
`conditions7. Digital PCR offers more accu-
`racy and less ambiguity than qPCR, he says,
`but qPCR has enticing advantages. It is less
`expensive and works over a much broader
`
`TATAA Biocenter
`
`
`
`
`
`Digital PCR works by diluting a sample into many partitions and counting up
`the number of partitions in which a reaction occurs.
`
`nature methods | VOL.9 NO.6 | JUNE 2012 | 541
`
`© 2012 Nature America, Inc. All rights reserved.
`
`npg
`
`MYR 1018
`Myriad Genetics, Inc. et al. (Petitioners) v. The Johns Hopkins University (Patent Owner)
`IPR For USPN 7,824,889
`
`Page 1 of 4
`
`

`

`technology feature
`
`table 1 | Commercial digital PCR offerings
`
`consumables and
`list price
`12 arrays per chipa (765 wells
`per array): $400 per chip
`(works in both EP1 and
`BioMark)
`48 arrays per chipa (770 wells
`per array): $800 per chip
`(works in both EP1 and
`BioMark)
`OpenArray platesa (64 holes
`per subarray): $150 per plate
`
`number and volume
`of partitions
`12-inlet chip: 9,180
`partitions, 6 nl per
`partition
`
`48-inlet chip: 36,960
`partitions, 0.85 nl per
`partition
`
`Varies; 3,072 partitions
`per plate, 48 subarrays per
`plate, 33 nl per partition
`(machines run 3–4 plates
`at once)
`
`Volumes required
`12-inlet chip:
`8 ml of mix, ~4 ml
`of sample; 57%
`analyzedb
`48-inlet chip:
`4 ml of mix, ~2 ml of
`sampleb
`
`100 ml of sample
`per plate (across 48
`arrays)
`
`qPcr
`capacity multiplexing
`Yes
`Can use up to 5 colors
`to detect 5 targets
`(assumes 5th color is
`ultraviolet)
`Can use up to 5 colors
`to detect 5 targets
`
`No
`
`Yes
`
`Uses 2 colors of probes
`to detect 2 targets
`
`Vendor
`
`Fluidigm
`Corporation
`
`Instruments
`and list price
`BioMark HD:
`$200,000–$250,000
`
`EP1:
`$100,000–$150,000
`
`OpenArray RealTime
`PCR System and
`QuantStudio 12K Flex
`instrument:
`$140,000 and
`$90,000–$190,000,
`respectively
`QX100 ddPCR System
`(machines to generate
`and read droplets):
`$89,000
`
`Life
`Technologies
`
`Bio-Rad
`Laboratories
`
`8 samples per chip
`(14,000–16,000 droplets per
`sample): $3 per sample
`
`Up to 9 ml per
`sample (20,000
`droplets made); an
`average of 70% read
`
`No
`
`Uses 2 colors to detect
`2 targets
`
`Up to 96 samples per run
`(assumes manual pipetting
`into PCR plate); 1,344,000
`partitions per run (assuming
`separate thermocycler runs
`12 chips at once), 1 nl per
`partition
`8 samples per run; up to
`80,000,000 partitions per
`run, 5 pl per partition
`
`RainDancec
`
`8 samples per chip (up to
`10,000,000 droplets per
`sample): $10–$30 per sample
`
`Uses 2 colors, but
`RainDrop Digital
`can use varying
`PCR (machines to
`concentrations of
`generate, collect
`probes to detect up to
`and read droplets):
`10 targets
`$100,000
`aArrays can hold separate samples, or the same sample can be spread over multiple arrays. bFor rare allele analysis, protocols are available to eliminate the dead volume. cPlans full commercial launch later this year.
`
`5–50 ml per sample
`
`No
`
`dynamic range than digital PCR. For exam-
`ple, it can determine that transcripts of one
`gene are as much as a billion times more
`abundant than transcripts of another gene.
`Also, qPCR experiments can routinely
`analyze hundreds of sequences per sam-
`ple run. Eventually, Kubista believes that
`it will be possible to multiplex dPCR to
`examine perhaps as many as 100 reac-
`tions at once, but no one would con-
`sider measuring large numbers with
`digital PCR today, he says. And qPCR
`is already well-integrated into many
`researchers’ labs. “We’ve been develop-
`
`ing workflows for qPCR for 20 years.”
`In contrast, the first full conference dedi-
`cated to applications of digital PCR is
`scheduled for October of this year (see
`http://www.healthtech.com/digital-pcr).
`MicroRNA res e archer Mune esh
`Tewari at the Fred Hutchinson Cancer
`Research Center uses digital PCR in situ-
`ations where absolute quantification is
`important, such as when detecting low-
`abundance RNA. One advantage of digital
`PCR is that with more partitions, a greater
`volume of a dilute RNA sample can be ana-
`lyzed, he says.
`
`Also, digital PCR does not require the
`calibration and internal controls neces-
`sary for qPCR. Instead, counts from rep-
`licate wells can simply be added together.
`“Thinking in terms of absolute copies is so
`intuitive,” he says. Nonetheless, his lab cur-
`rently performs more qPCR experiments
`than digital PCR experiments. qPCR has
`lower cost and higher throughput, says
`Tewari, and his staff is more familiar with it.
`That’s a typical situation, says Paul
`Pickering, head of the digital PCR business
`unit at Life Technologies. “Most customers
`are seeking to do RT-PCR and then, in the
`situations that they need it, they’ll deploy
`digital PCR.” In those cases, he says, “there
`are four attributes that customers value:
`sensitivity, specificity, precision of the
`answer and the fact that you can get an
`absolute count without needing to refer-
`ence any other material.”
`
`digital Pcr on chips
`In 2006, Fluidigm became the first com-
`pany to commercialize digital PCR. It
`offers two systems that mix samples with
`reagents, partition the reaction mix-
`ture, perform thermocycling and read
`
`Fluidigm
`
`Fluidigm Corporation’s BioMark HD System for digital PCR and qPCR.
`
`542 | VOL.9 NO.6 | JUNE 2012 | nature methods
`
`© 2012 Nature America, Inc. All rights reserved.
`
`npg
`
`Page 2 of 4
`
`

`

`technology feature
`
`to a droplet reading machine, which func-
`tions like a flow cytometer to analyze each
`droplet for whether or not a reaction has
`occurred.
`QuantaLife launched the first com-
`mercial digital droplet PCR system a year
`ago. In December 2011, the company was
`acquired by Bio-Rad Laboratories for $162
`million, with promises for more cash if
`products hit certain milestones. All along,
`the goal was to develop an instrument that
`was both inexpensive and easy to use, says
`Bio-Rad marketing manager Mike Lucero,
`who was an early employee of QuantaLife.
`“We have two rules at the company: no
`chips and no lasers.” He’s betting that the
`low cost of consumables will set the com-
`pany apart, he explains, holding up a clear,
`lightweight strip studded with sets of cups
`for holding collections of droplets, each
`narrower than a toothpaste cap. “This is
`less than $10,” he says. “And it’s because
`we took the time and effort to make it out
`of plastic.”
`Getting the chemistry for the droplets
`right was crucial, says Ben Hindson, one of
`QuantaLife’s original employees and now a
`senior principal scientist at Bio-Rad. The
`droplets produced must remain a uniform
`size even if the temperature fluctuates
`slightly as they are generated. What’s more,
`the droplets cannot burst or coalesce dur-
`ing handling, thermocycling and reading,
`and they also must maintain biocompat-
`ible conditions that support PCR. It takes
`25 minutes to generate droplets for 96
`samples, says Hindson, and one person
`running the system can analyze 3 sets of 96
`samples a day. The technology has begun
`to appear in independent research; in a
`high-profile paper combining genomic,
`transcriptomic, proteomic and metabolo-
`mic data, digital droplet PCR developed by
`QuantaLife was used to detect differential
`expression of a variety of alleles1.
`Another digital PCR system has been
`developed by RainDance and is scheduled
`to launch later this year. The machines in
`
`RainDance
`
`RainDrop Source and RainDrop Sense machines
`for droplet digital PCR.
`
`nature methods | VOL.9 NO.6 | JUNE 2012 | 543
`
`Life Technologies
`
`Life Technologies’ QuantStudio System for digital
`PCR and qPCR.
`
`we found is that a lot of our customers
`want the capability [for digital PCR] but
`aren’t ready to jump in and say that that’s
`the only thing that they have to do,” says
`Pickering. Buying a machine that can do
`both, he says, is similar to the decision to
`purchase a hybrid gas and electric automo-
`bile rather than an electric-only vehicle.
`
`digital Pcr in droplets
`Companies like Bio-Rad and RainDance
`sell machines that cannot perform qPCR
`but which offer many more partitions. In
`droplet digital PCR, reaction chambers are
`separated not by the walls of a well but by
`carefully titrated emulsions of oil, water
`and stabilizing chemicals. First, samples
`are put into a machine where they are
`mixed with all the necessary reagents and
`dispersed into tiny droplets. The droplets
`for each sample are transferred into tubes
`that can be placed in a thermocycler for
`PCR. Afterward, the tubes are transferred
`
`results within each partition. The simpler,
`cheaper EP1 machine detects only end-
`points, that is, whether or not a reaction
`has occurred. The BioMark HD System,
`which also performs qPCR, can be set to
`monitor the course of a reaction and pro-
`vide data that can eliminate false positives.
`Both systems use chips containing sophis-
`ticated microfluidics and tiny valves that
`partition samples into about 800 reactions,
`with either 12 or 48 samples per chip. If
`researchers want to run more reactions
`per sample, they can just double up arrays
`within chips or even double up on chips,
`says Ramakrishnan.
`The company has developed another
`chip called the 200K with hundreds of
`thousands of partitions, and has licensed
`separate technology for chips with as
`many as a million partitions. However,
`plans to commercialize these technologies
`are on hold pending greater demand. “We
`can go up in terms of partitions, but we
`haven’t found a huge pull from the mar-
`ket in going to that higher density,” says
`Ramakrishnan.
`Life Technologies began offering digi-
`tal PCR in 2009 after acquiring long-time
`collaborator BioTrove. It now sells two
`machines that can be used for both digi-
`tal PCR and qPCR, the OpenArray and
`QuantStudio 12K Flex. These mix samples
`with reagents, load mixtures into reac-
`tion chambers, run amplification cycles
`and monitor reactions as they occur. The
`machines rely on plates that are roughly
`the size of a microscope slide and are
`essentially highly engineered peg boards
`with nano-sized holes; capillary forces
`and careful placement of hydrophilic and
`hydrophobic surfaces hold samples in
`place.
`The OpenArray machine holds up to
`three plates, each containing 48 arrays
`with 64 partitions apiece. QuantStudio
`holds up to four plates and can also accept
`formats used in high-throughput qPCR
`experiments: TaqMan Array cards as
`well as 96- and 384-plate formats. “What
`
`Bio-Rad
`
`Bio-Rad’s QX100 droplet digital PCR System.
`
`© 2012 Nature America, Inc. All rights reserved.
`
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`Page 3 of 4
`
`

`

`technology feature
`
`The most common
`applications of
`digital PCR at the
`TATAA Biocenter are
`standardizing qPCR
`assays, detecting
`copy number
`variations, detecting
`rare mutations
`and distinguishing
`differences between
`expression of nearly
`identical alleles, says
`Mikael Kubista.
`
`this system gener-
`ate and read mil-
`lions of picoliter-
`sized droplets, a
`feature that not
`only allows sci-
`entists to identify
`rarer alleles but
`also alleviates some
`of the need to dilute
`samples carefully.
`“With all those
`droplets, we can
`deal with a wide
`variety of different
`concentrations,”
`explains company
`co-founder Darren
`Link. He dismisses
`competitors’ claims
`that millions of
`partitions are more
`than most scien-
`tists will need. “Too
`many is never a problem, especially when
`you are talking about expression analysis,”
`he says. “You don’t want to run titrations to
`find the sweet spot of the dynamic range.”
`Link also emphasizes that the system does
`not require any manual pipetting as drop-
`lets are moved between machines that make
`droplets, perform thermocycling and ana-
`lyze droplets.
`Researchers at RainDance and the
`University of Strasbourg and University
`Paris-Descartes reported that they could
`detect one mutant KRAS gene within
`200,000 wild-type KRAS genes when the
`former was diluted into genomic DNA. The
`seven most common KRAS mutations were
`screened in two multiplex experiments:
`one examining the wild-type allele along
`with four mutations and one with the wild
`type alongside three mutations8. At AACR,
`RainDance presented results detecting can-
`cer mutations in patient serum.
`
`thinking digitally
`Digital PCR may not require the same kind
`of calibration and controls as qPCR, but
`there is still plenty of scope for artifacts,
`says Huggett. Working in tiny volumes
`and with single-molecule concentrations
`is a complicated engineering feat. “dPCR
`is at an early stage, so my advice would be
`to proceed with caution and be careful of
`sweeping statements,” he says. For exam-
`ple, some researchers believe that digital
`PCR will be less susceptible than qPCR to
`
`544 | VOL.9 NO.6 | JUNE 2012 | nature methods
`
`enzyme-inhibiting substances that occur
`in some samples. For qPCR, the problem
`is that inhibitors increase the number of
`amplification cycles required to reach a
`given signal. But even though digital PCR
`does not count cycles, inhibitors could still
`be a problem if they cause false negatives by
`preventing reactions from occurring at all.
`Some factors are particularly impor-
`tant to consider with digital PCR, says
`Kubista. “For example, it is really critical
`that the assay is well-performing, that you
`are confident that if there is a single target
`molecule [you] will see it. Not all assays
`are that good.” In addition, researchers
`need to make sure that multiple sequences
`of interest do not
`appear on the same
`piece of DNA; oth-
`erwise, they can-
`not be separated
`into different par-
`titions. (Also, if
`the positive parti-
`tions are clustered
`toget her rat her
`t h a n r a n d o m l y
`dispersed, there is
`probably an issue
`with sample load-
`ing or analysis.)
`Sp e c i f i c it y i s
`also an issue. Many
`assays will amplify
`pro du c t s ot h e r
`than the sequence
`of interest, particularly if pseudogenes
`are present. Understanding rates of false
`positives is crucial when hunting for rare
`alleles. In these cases, most partitions will
`not contain the molecule of interest, and
`the number of false positives could dwarf
`the number of true positives. For these
`reasons, Kubista recommends a variety of
`control experiments. His center offers a kit
`called ValidPrime that amplifies just one
`copy of a gene per haploid genome and can
`be used to assess specificity.
`Special consideration is warranted for
`the rarest alleles. If a sequence is only
`going to occur once in 50 microliters, says
`Pickering, it’s essential to analyze more
`than 50 microliters of the sample. “No
`matter what the technique, if you haven’t
`sampled enough volume to get what you’re
`looking for, you’re not going to detect it.”
`In applications for quantifying more
`abundant molecules, such as detecting
`copy number variants or measuring gene
`
`“At the moment,”
`says Jim Huggett,
`“digital PCR is a
`specialist approach
`that is much more
`costly than qPCR, and
`qPCR is suitable for
`the vast majority of
`applications.”
`
`expression, researchers generally need
`to get a rough estimate of the concentra-
`tion of their target of interest in order to
`make appropriate dilutions. Otherwise,
`too many partitions will contain multiple
`copies. (Statistics can compensate for this,
`but only to a limited extent.) If every parti-
`tion shows a reaction, researchers cannot
`calculate the concentration of the original
`molecules, explains Kubista. “You get the
`best use of the chip by having 80% posi-
`tive [partitions]. If the number rises above
`90%, precision drops.”
`Monitoring the ‘response curve’, or how
`levels of DNA change over the course of
`amplification, can help eliminate false read-
`ings caused by nonspecific labeling of DNA
`sequences—a benefit that companies such
`as Fluidigm and Life Technologies, which
`provide such data, are keen to point out.
`Advocates of droplet digital PCR, however,
`believe that accurate measurements can be
`made with endpoint data alone, and cite the
`advantages of a greater number of parti-
`tions. “For allele-specific experiments, you
`may get false positives, but you can quan-
`titate what that false positive rate is rather
`than infer from a curve,” says Hindson.
`Researchers should also consider all the
`steps that occur before digital PCR begins,
`says Kubista. As samples are processed,
`material is lost. Running controls in which
`a sequence is spiked in before processing
`can help determine how much sample is
`necessary, he says.
`Although the experts urge care in
`designing digital PCR experiments, they
`are enthusiastic about its potential. As the
`technology matures and the costs come
`down, more researchers will learn to ask
`questions only digital PCR can answer,
`says Kubista. “There are a few applications
`today, and there will be more tomorrow.”
`
`Monya Baker is technology editor for
`Nature and Nature Methods
`(m.baker@us.nature.com).
`
`1. Chen, R. et al. Cell 148, 1293–1307 (2012).
`2. Tadmor, A.D., Ottesen, E.A., Leadbetter, J.R. &
`Phillips, R. Science 333, 58–62 (2011).
`3. Wang, J. et al. Clin. Chem. 56, 623–632 (2010).
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`8. Pekin, D. et al. Lab Chip 11, 2156–2166 (2011).
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`© 2012 Nature America, Inc. All rights reserved.
`
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