PCR is a workhorse of biotech—whether it’s being used to prepare samples for another assay, or to assay samples from another preparation. From the search for point mutations to discovery of methylated DNA biomarkers, homebrew tests can be crafted to fill in where off-the-shelf kits fall short.
Real-time (q)PCR gives quantitative readings while end-point PCR affords digital answers, which can themselves be quantitative. Researchers at Select Sciences’ “qPCR Europe Conference”, held in Munich last month, powwowed about what uses they put it to in their search for more reliable, sensitive, automatable, and innovative diagnostics.
Ready-to-use kits are available for PCR-based diagnosis of many, if not most, of the viral infections that routinely find their way into the clinic. Yet when it comes to highly pathogenic viruses like pox viruses—which, because they can quickly kill off their hosts, tend to be far less common—“there’s definitely no market,” noted Andreas Nitsche, Ph.D., of the Center for Biological Safety 1 at the Robert Koch Institute in Berlin.
To identify such pathogens “you first need your own assays—your own assay design, your own controls, your own procedures how to establish such an assay,” he explained. “It’s not like a routine clinical lab.” The generic infectious agent needs to be found, and then the species or variant identified. For this, the pox expert says, bioinformatics plays an increasingly important role.
Viruses like the pox viruses have a very conserved genome, and their sequences can be compared by eye or with standard software tools. But in cases involving more heterogeneous viruses like the flaviviruses, dengue, or yellow fever, “we are talking about many, many thousands of sequences, and you have new sequence entries in GenBank almost every week,” he pointed out.
Assays need to be checked to make sure they’re still valid for all the known variants. And aligning and comparing thousands of sequences can take weeks or months. Dr. Nitsche is currently testing a plug-in for commercial software—to be released soon—that promises to streamline the process by providing automatic tools for sequence control and theoretical validation of assays.
Even identifying a particular virus type—for example, that the etiological agent of SARS is a coronavirus—can also be a challenge. This had been done with electron microscopy (EM), which is “the perfect open-view tool—you see everything that is in the sample,” Dr. Nitsche said.
But EM has its limitations: it will only identify the family of pathogen (it can’t discriminate between smallpox and vaccinia, for example), and it requires a relatively large pathogen concentration (in the range of 105-6) that may not be found in blood or other infected body parts.
Dr. Nitsche and other virus hunters have been exploiting techniques such as generic PCR—using generic primers that will amplify everything in the sample—as well as deep sequencing to find sequences that may not have been known before. Then, “you have a real need for bioinformatics because you have to handle a ton of data.”
At present, a qualitative nucleic acid-based test is all that is needed to diagnose the presence of a pathogen, said Philip Day, Ph.D., reader in quantitative analytical genomics at Manchester University—it’s there or it’s not. But when looking for biomarkers for cancers and the like, on the other hand, it’s important to get a measure of how much is there, and on how many cells.
Yet these tests fail to take heterogeneity into account. “They tend to involve ‘bulk analysis’, whereby one starts off with these tissues with lovely architecture and structure, and then we jump in and lyse these tissues for a supernatant of nucleic acids, from which we try and ascertain some sort of diagnostic value via qPCR,” Dr. Day said.
qPCR is avidly good at picking up an aberration such as a tumor marker in a biopsy or blood sample. The problem is that it’s very hard to know the number of cells actually carrying the aberration and the distribution of aberrations across the cancer population. In addition, qPCR can be impeded if the target nucleic acids are too heavily diluted by other, nontarget nucleic acids.
As diagnosis and therapy move closer to the goal of personalized medicine, risk groups need to be more precisely defined by more subtle measurements of biomarkers. Dr. Day is aiming toward single-cell analyses, calculating answers in numbers of molecules per cell. For now that means employing homogeneous extractions commencing with FACS to sort individual cells into 96-well plates for qPCR.
Ultimately he hopes to be able to do the same thing using a high-throughput, continuous flow microfluidics system with integrated two-phase PCR and fluorescent detection. Since this is the result of a single cell, it’s not always necessary to use real-time optics, Dr. Day explained.
Yet the emphasis on single cells puts him in a strange predicament: data from a single cell isn’t necessarily indicative of the disease or tissue, Dr. Day confessed.
“So therefore what we’re doing presently is trying to ramp up the numbers of individual cells that we’re analyzing, to identify what is the minimum number of single cells we need to analyze by amplification, which will give us a full portrayal of the population of cells.” This, in turn, will be used to colonize databases to make full sense of the single-cell information in the context of disease progression and treatment.
Back to the Future
Biomarkers are often about more than just how the As, Gs, Ts, and Cs line up. Much work of late has concentrated on epigenetic DNA modifications such as methylation of CpG dinucleotides, which can have profound implications for the expression of tumor suppressors and other genes.
Frequently the search for methylated DNA starts with bisulfite conversion of unmethylated cytosines to uracils (leaving methylated cytosines untouched), after which a variety of techniques involving standard or methylation-specific PCR (MSP) can be used. Collectively these are considered bisulfite sequencing.
Andreas Weinhäusel, Ph.D., senior scientist at the Austrian Institute of Technology, was using bisulfite deamination-based MSP to look for differentially methylated genes in leukemia. The conversion process itself fragments and degrades the DNA, and so there is a tradeoff between the specificity that results from higher stringency and longer incubation times, and the sensitivity of detecting unmethylated vs. methylated DNA found under less stringent conditions.
What the proper balance is “changes also from gene to gene,” he noted, making it less than ideal for the genome-wide screening and validation of already identified markers he was undertaking.
Dr. Weinhäusel decided to return to the “more or less forgotten” technique of using methylation-sensitive restriction enzymes (MSREs) in place of bisulfite conversion as a precursor to qPCR to examine the methylation status of genetic sequences. “Everybody used restriction enzymes over the last 30–40 years,” he said.
The number and quality of the enzymes and the conditions for handling them have improved, making him predict that they “might have some sort of renaissance in qPCR and methylation testing.” MSRE-qPCR validated markers from their own studies on lung cancer were confirmed by bisulfite-pyrosequencing resulting in almost perfect classification of tumors and normal tissues by both methods.
There are several other advantages of MSRE-based analysis over bisulfite-based approaches, he noted. It’s easier to design PCR primers for native than for converted DNA; multiplexing and parallel analyses are simpler; less template is required; sequence information can be lost with bisulfite conversion; and because there is no deamination, no purification is required—and fewer steps means less chance of introducing bias.
There may be an issue in designing PCR conditions for native DNA from CpG islands, such as Fragile X syndrome’s ccg repeat and other C-rich repeat expansion diseases: the very high CG content means the DNA melts at higher temperatures—often as high as 95–100°C. “In that situation I think it is really useful to have the bisulfite conversion because here you are dramatically reducing the Tm of that target region,” he explained.