May 2013

Let’s get digital

As PCR becomes faster, more multipurpose and
more precise, a handful of researchers are using a
digital approach to find a needle in a haystack.

Thirty years ago, Kary Mullis and his team at Cetus Corp. were the first to amplify short sequences of DNA in vitro. Dubbed the polymerase chain reaction, this technology involves taking a template piece of DNA, adding excess amounts of free nucleotides along with short sequences to prime the ends of the DNA molecules and specify the region of amplification, and letting a DNA polymerase copy the template during multiple amplification cycles. Their idea revolutionized modern science and now is used across the fields of forensic science, astrobiology, cancer biology and many others.
“It’s amazing that one simple thing spread,” says Mullis. “There are not a lot of areas of biochemistry that haven’t benefitted” from PCR. The Nobel selection committee realized the impact of PCR, and Mullis was recognized for its invention with a Nobel Prize in chemistry in 1993.
“PCR has pretty much single-handedly catapulted molecular biological methodology into being a key part of almost every aspect of biological and clinical research,” says Jim Huggett of the National Measurement Institute for Molecular Biology and Biochemistry in the United Kingdom. “From vaccine development to metegenomics, the simplicity and versatility of PCR has been crucial in developing many fields.”
The technology of PCR has undergone revolutionary changes since its introduction in 1983. PCR is now faster, more multipurpose and more precise. The latest offshoot uses PCR to count single copies of a particular DNA sequence present in a sample. This technique, called digital PCR, can precisely quantify the amount of a given sequence of DNA among a complex mixture of sequences and express that quantity as a numerical value. 
Digital PCR is related to the technique known as quantitative PCR, but it takes the quantification of DNA one step further. Quantitative PCR can compare the amount of a particular sequence to a reference sequence, one that has a known or standard amount across all samples. Because of this, quantitative PCR is actually an analog technology; the measurement uses an analog between the sample of interest and the reference sequence to make the quantification. Digital PCR does not require a reference or standard, making the quantification of DNA more precise than what quantitative PCR can achieve.
“I think digital PCR has a lot of possibility, and the digital aspect is very cool,” says Rob Phillips of the California Institute of Technology. “There are many, many ways you can imagine using it.”

Kary Mullis recalls the moment he conceptualized PCR

Kary MullisKary Mullis originally conceived the idea for polymerase chain reaction while was working through the problem of how to diagnose sickle cell disease in a shorter amount of time. The existing method took three months from testing to diagnosis, so Mullis tried to apply his expertise in working with DNA oligonucleotides to develop a faster assay that could be done using clinical samples.
Sickle cell disease is caused by a single base-pair change in the DNA, so Mullis’ strategy was to do a dideoxy sequencing reaction at that specific base pair in the genome, directed to the location by oligos designed to the surrounding sequence. Performing the reaction on both strands of the DNA would ensure accuracy.
The main obstacle, Mullis thought, was the presence of endogenous deoxynucleotides (dNTPs) in the clinical samples. These dNTPs would interfere with his sequencing reaction, because a polymerase would use them preferentially over dideoxynucleotides (ddNTPs), the basis of the sequencing technique he was using. His solution for rapidly depleting the dNTPs from the sample? Add a polymerase. The polymerase would use all the dNTPs, leaving the sample ready to be prepared for sequencing.
Mullis recounted, “I was driving to my cabin while I was thinking about this, and it was about one mile into the drive when I realized: This is going to copy both sides [of the genomic sequence], and I’ll end up with two times the signal than before!” He realized the polymerase would use the dNTPs to make an exact copy of the target region, and the amount of target would be doubled. He thought, “I could do this as many times as needed!”
“Once I had the picture of that in my head,” Mullis said, “I slammed on my brakes. I was in the middle of a busy highway, and I thought, ‘I better get off the road before I get killed!’ I pulled over, and I was just stunned. I thought, ‘210 was 1,024. 220 was over 1 million. I could make a million copies of the target.’ Easy.”
Back in the lab, Mullis industriously worked on his new technology, which he would later call polymerase chain reaction, after the file he had created on his computer (chain_reaction.pol). Although he could only stand to manually perform 10 reaction cycles, he successfully amplified plasmid DNA to an amount visible on an ethidium bromide-stained agarose gel. His idea, as he simply put, “worked, beautifully.”

Quantifying exact number of copies
Digital PCR involves diluting a DNA sample and placing the template DNA into micro-wells before performing hundreds or thousands of reactions with the same source material. The sample is diluted to the point at which either zero copies are contained or one copy of the template is contained in each reaction. Therefore, the reaction readout will be either positive (containing the template) or negative.
By comparing the ratio of positive to negative reactions and taking into account the dilution factor, digital PCR quantifies exactly how many molecules of the template were present in the original sample. This method is low-throughput by nature because one must run so many reactions on each individual sample, but overall it is considered more sensitive and more precise than quantitative PCR.
Several platforms exist for performing digital PCR, all involving minute reaction volumes. Emulsion in tiny droplets, microfluidics-based chips and hydrophobic/hydrophilic chambers all have been used for digital PCR. Companies specializing in these technologies, BioRad, Fluidigm and Life Technologies, have tried to optimize their systems to include features such as a large reaction number or the ability to perform quantitative PCR while the reaction cycles and then using the endpoint of the reaction to generate the digital PCR results.
Real-time PCR provides a curve or graph that measures the amplification of DNA in a reaction at the end of each PCR cycle. The real-time data for a single well of a digital PCR reaction can help determine if a detected target sequence is real as opposed to a false positive or contaminant. The readout for digital PCR requires only the endpoint value, answering the question, “Did the reaction amplify DNA or not?”
For some digital PCR platforms, real-time data is not collected as the machine cycles. This means that the endpoint value is still useful for obtaining digital PCR results, but there is no way to know whether a positive reaction is a false positive. The advantage of such a system is that amplification is more rapid and can be formatted to run a higher number of reactions at once. The more reactions that can be done, the more sensitive the quantification becomes. Sensitivity depends on the volume screened, so performing more reactions equals screening a larger volume. Also, running more reactions improves precision, because more replicates can be run to determine the consistency of data between reactions. 
Versatility of digital PCR
The uptake of the digital PCR technology by various research groups has been on the rise.
A search of the term “digital PCR” on PubMed shows that 14 publications recorded the use of the technique between 1999 and 2009. In 2010 alone, 15 publications did. This number stays fairly constant until 2013: Already, 13 publications are listed, which puts digital PCR-related publications on track to hit 52 by the end of the year, a three-fold increase over 2012.
Digital PCR is being used by regulatory agencies in the United Kingdom as the new standard in quantifying DNA. In a study published last year in the journal Nucleic Acids Research by Alexandra Whale and colleagues at LGC, the UK’s designated National Measurement Institute for chemical and bioanalytical measurement, microfluidic digital PCR was compared with conventional quantitative PCR in the measurement of copy number variation, or CNV, associated with tumors.
By measuring the CNV of the HER2 gene, the group showed that digital PCR could measure reliably a smaller CNV than quantitative PCR could. The group chose to use digital PCR, because it could measure the exact number of CNV sequences and because it did not require the use of a calibration curve.
Moving from quantitative PCR to digital PCR presents some difficulties. Jim Huggett, an author on the HER2 study, had a few concerns. “At the moment, cost has to be mentioned. But technically, the most challenging is the low dynamic range of the instrument,” he said. “Another big challenge is total reaction volume — and particularly the amount of sample you can get into a reaction. Volume for volume, digital PCR may be more accurate than quantitative PCR, but if you can only get 1 μl into a digital PCR reaction, as opposed to 20 μl into a qPCR reaction, then this will reduce the physical sensitivity.”
Huggett said he hopes his team’s publication showcases the method and the way it improves the quantitative measure of DNA. He says he sees digital PCR as a technique with the potential to considerably reduce variability between experiments done in different labs, improving the reproducibility of experiments.
“Most measurement techniques used with biological measurement are relative (such as qPCR or ELISA) and require a calibration curve to assign a value. Digital PCR does not need a calibration curve, because the results are digitized and surprisingly reproducible,” says Huggett.
Digital PCR also is providing a platform to address broader questions in microbiology. In their paper “Probing individual environmental bacteria for viruses by using microfluidic digital PCR,” Arbel Tadmor and colleagues at the California Institute of Technology demonstrated a creative use of digital PCR. Published last year in the journal Science, the study looked at viruses infecting bacterial cells in the termite hindgut. Although bacteriophage and other viruses are commonly found, specific virus-host interactions remain unknown due to problems with obtaining cultures from the environment.
Culturing the bacterial hosts before isolating and identifying the viruses is tedious work and can be ineffective at times. More than 99 percent of microbes cannot be cultured in a lab.
Tadmor and colleagues decided to use digital PCR to identify the viruses present in an individual bacterium from an environmental sample.
Having a lab space next to Jared Ledbetter, a colleague of Steve Quake (founder of the Fluidigm digital PCR platform), made using digital PCR a natural choice for Tadmor. “The Fluidigm product that Jared was using was this digital PCR chip,” he says. After a member of Ledbetter’s lab had success using digital PCR to identify functional genes present in microbes, Tadmor’s adviser, Rob Phillips, wanted to try something similar.
“Rob, whose lab, among other things, works on bacterial viruses, thought, hmm, this would be cool to try to pair viral genes with the identity of the host. The reason that’s an interesting problem is that most of the hosts in the microbial world cannot be cultured, and if you can’t culture either the host or the virus, then you can’t really know who is infecting whom in the microbial world,” Tadmor explains.
By diluting their samples so that analysis could be carried out on the single-cell level, they were able to identify which viruses infected which bacteria. They multiplexed two templates in each digital PCR reaction — one to recognize a viral marker and one to recognize the bacterial 16S rRNA gene.
Tadmor, who performed the digital PCR in the study during his time as a graduate student, says he encountered unique challenges while working with viral genomes. He emphasized the difficulty of using PCR to detect ever-mutating viral genomes as well as extracting the reaction components from the physical microfluidic chips for further analysis. “It’s a general problem with the method to get the (PCR) chemistry to work. (It’s) really hard to get a multiplex qPCR reaction to work on the chip,” he says.
Barring these troubles, however, Tadmor said he hopes that his paper will inspire others studying virus-host interactions to use this technology to ask similar questions. 
“That’s something which in the past has been difficult to tackle,” he explains.
Over all, digital PCR has garnered excitement, but the size of its impression on the scientific community is yet to be determined. One indisputable fact, however, is the legacy of traditional PCR.
“PCR has had the same sort of impact on the world of biology that the telescope had on astronomy: It’s huge,” says Phillips of Caltech. “It’s really hard to find any domain of biology that was not touched by PCR. In 30 years, it’s amazing that we’ve come as far as we have.”

Diedre RibbensDiedre Ribbens ( is a graduate student at the Johns Hopkins School of Medicine and a budding science writer. Connect with her on LinkedIn.

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