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Feature Articles : Apr 1, 2013 ( )
Development and Evolution of PCR
In the six decades since Watson and Crick identified the double helical structure of DNA, a series of elegant and powerful experiments has established the broad outlines of how genetic information is encoded, replicated, and expressed.
In the 1980s, the development of the polymerase chain reaction (PCR) dramatically simplified access to genomic information, facilitating both basic research studies and a wide variety of applications ranging from clinical diagnostics to forensic analyses.
PCR—the enzymatic amplification of a specific DNA fragment targeted by two oligonucleotide primers—is a surprisingly simple concept but a technology that has become increasingly powerful and broadly implemented. Starting with a DNA or RNA template, repeated cycles of denaturation, primer annealing, and polymerase-mediated primer extension generate an exponential accumulation of a specifc targeted fragment that can be analyzed by a variety of methods.
It is now difficult to conceive of any nucleic acid based study that does not incorporate PCR in some form in some stage of the experimental protocol.
In the ’80s, researchers explored a variety of signal or probe amplification systems for research and diagnostic applications. In contrast, PCR, with its sequential cycles of targeted oligonucleotide primed synthesis, is a target-amplification system, capable of amplifying unknown genomic regions between the primer sites.
While sequence information is necessary for primer design, some mismatches between primer and template, primarily those at the 5´ end of the primer, could be tolerated. As a result, primers based on a sequence from one species could be used to amplify the homologous gene from related species, or those from one gene in a multigene family could amplify related genes.
In addition, valuable sequence information, such as restriction sites to facilitate cloning or, more recently, adaptor and sample barcode sequences for next-generation sequencing (NGS), could be introduced at the ends of the amplicon via tolerated mismatches at the 5´ end of the primer. Labels could also be introduced into the amplicon using primers labeled at the 5´ end, facilitating both gel electrophoresis and probe hybridization analyses.
PCR could also be used to confer on any DNA fragment the capacity to be replicated by simply ligating primer sites onto the ends of the targeted fragment(s), a property currently used in the preparation of shotgun libraries for NGS.
PCR is essentially agnostic with respect to analytic methods: gel electrophoresis, probe hybridization, Tm melting analysis, sequencing, and a variety of other techniques have all been successfully applied to the analysis of amplicons.
PCR’s ability to generate high concentrations of a specific labeled DNA target created a paradigm shift in oligonucleotide probe hybridization analysis. Previously, target DNA had been immobilized on a substrate, denatured, and hybridized under stringent conditions (so that a single mismatched base-pair would destablilize the target-probe duplex) to a vast molar excess of labeled probe (the “dot blot”).
With PCR, a panel of sequence-specific oligonucleotide probes could be immobilized and then hybridized to a labeled amplicon (“reverse dot blot”). This basic principle became the basis of the linear array, probe-coupled beads, and microarray analysis, in which oligonucleotide probes are immobilized (by synthesis or deposition) on a membrane, bead, or chip.
Evolution of PCR Technology
The three decades since the first PCR papers were published have seen many improvements, refinements, and modifications that have dramatically increased its analytic capabilities. However, the most transformative has surely been the introduction of a thermostable DNA polymerase.
In the first PCR experiments designed to amplify β-globin from human DNA, we used the Klenow fragment of E. coli DNA polymerase. This system required adding fresh enzyme after the heat denaturation step of every PCR cycle, a cumbersome procedure (automated, during its brief reign, by a Rube Goldberg–like machine, known affectionately as Mr. Cycle). Because the primers were annealed and extended at 37°C, nontarget regions were often amplified along with the target.
Under these conditions, the first successful genomic PCRs resulted in dramatic amplification and enrichment of the targeted 110 bp fragment of β-globin, but only around 1% of the amplified DNA was the target.
The introduction of the first thermostable polymerase in PCR (Taq polymerase, isolated from Thermus aquaticus) eliminated the need to add enzyme during each cycle, allowing the development of simple thermal cycling instruments to automate PCR. By allowing primer annealing and extension at elevated temperatures, the use of a thermostable polymerase also greatly increased the specificity of target amplification.
Since the introduction of Taq polymerase, many different polymerases from a variety of thermophilic bacteria and hyperthermophilic archaea have been isolated, characterized, and used in PCR. Some have enhanced 3´ exonuclease activity (proofreading) and, thus, increased fidelity. Others have the ability to use RNA as a template.
Discovery of DNA polymerases that could synthesize a cDNA strand from an RNA template made possible PCR assays for gene expression as well as detection and characterization of RNA viruses. Other polymerases have been “engineered” for particular properties by mutating specific functional sites so that, for example, dideoxyribonucleotides or ribonucleotides can be incorporated during the primer extension step. Other engineered polymerases are more discriminating in the extension of primers mismatched at the 3´ end, increasing the specificity of “allele-specific” PCR.
The specificity of PCR has been further enhanced by chemically modified polymerases (“hot start” PCR) that remain inactive at lower temperatures and only extend primers at elevated temperatures. An alternative method of ensuring that polymerases are inactive at lower temperatures is the use of “aptamers,” small oligonucleotides that have been selected in vitro to bind to and inhibit a specific polymerase but that unfold at higher temperatures, releasing an active polymerase.
These specificity-enhanced polymerases—along with primers that have been chemically modified at the 3´ end to prevent extension at lower temperatures—have been critical in achieving the sensitivity and specificity required by many clinical diagnostic applications.
Another significant step in PCR evolution was the development of real-time PCR growth curve analysis, based on monitoring the exponential accumulation of PCR product. Initially, the intensity of fluorescence generated by a dye bound to the double-stranded DNA amplicon was measured at every cycle. The shape of the growth curve and, in particular, the cycle at which the fluorescence intensity crossed a threshold, reflected the amount of target template in the reaction, allowing the development of quantitative homogenous assays. Then thermal cycling instruments with optics capable of measuring fluorescence at every cycle made real-time PCR the preferred method of quantitative analysis.
The incorporation of a probe, such as the TaqMan cleavage probe, added to the specificity of real-time PCR analyses and is now the basis of many qualitative as well as quantitative homogenous diagnostic tests. The TaqMan chemistry is based on the polymerase’s 5´ nuclease activity.
As the primer is extended, the polymerase can cleave an annealed oligonucleotide (labeled with a reporter and quencher), releasing a 5´ fragment in which the reporter, now separated from the quencher, generates a fluorescent signal. (The name, TaqMan, refers not only to the initial DNA polymerase used in this assay but also to the PacMan video game popular during the ’80s.) Other probe chemistries, such as fluorescent energy transfer between two annealed oligonucleotides, have also been used in real-time PCR.
Recently, the development of digital PCR, a system of massively parallel PCRs, each initiated with either one or zero target molecules, has made quantitative PCR analyses even more precise. The separation of the parallel PCRs can be achieved either by a microfluidics device (individual PCRs in individual chambers) or by an emulsion (individual PCRs in individual microdroplets).
Limiting dilution ensures that the individual reactions are initiated with a single target molecule and the number of positive PCRs can be counted to provide absolute quantitation. Like the allele-specific PCR systems used in many diagnostic tests, digital PCR is highly sensitive and is capable of detecting rare variants, such as cancer-related mutations (in oncogenes and tumor suppressor genes) in the presence of a vast excess of wild-type DNA.
The model system we used for the development of PCR was amplification from human genomic DNA of a β-globin fragment containing the codon 6 mutated in sickle cell anemia (SCA). So, the first demonstration of the diagnostic potential of this new technology was the prenatal diagnosis of SCA and, slightly later, for hemoglobinopathies in general via the detection of a large panel of β-globin mutations.
The same probe-based genotyping technology was used to develop an HLA typing test employed in the first forensic DNA test in 1986, and in 1991, in the first commercial PCR assay, the HLA-DQ-alpha Forensics test. Widely used during the ’90s, this forensics test of the highly polymorphic HLA-DQA1 locus has now been replaced with a panel of STR (short tandem repeat) markers.
Since our first case (Pennsylvania v. Pestinikis), PCR genetic typing has transformed the criminal justice system, helping convict the guilty and exonerate the wrongly convicted. These forensics genetic markers have also been used to identify missing persons and the victims of mass disasters, and for clinical diagnostic analyses of mixtures such as post stem cell transplant engraftment monitoring.
In the HLA field, serologic HLA typing, previously used for matching transplant donor and recipients as well as disease association studies, has been replaced by PCR-based typing.
The first clinical molecular diagnostics in-vitro diagnostic (IVD) test was a PCR-and-probe based assay for Chlamydia trachomatis. Arguably, the PCR diagnostic test that had the most clinical impact was the HIV viral load test. Introduced during the height of the AIDS epidemic, this assay not only allowed monitoring HIV patients’ disease but also provided a surrogate marker for testing new antiviral drugs, measuring their effect on viral load rather than by following clinical progression (slower and more subjective).
Today, most quantitative tests are based on the real-time PCR quantitative analysis with TaqMan probes. PCR-based multiplex tests for HIV, HCV, and HBV are also widely used in most blood screening programs.
Another PCR-based viral assay with a significant impact on clinical practice involves detection of human papilloma virus (HPV) in cervical swabs. PCR was instrumental in demonstrating that HPV is the cause of cervical cancer, and PCR assays—particularly those capable of distinguishing the very high-risk HPV16 from the other high-risk HPV types—have been shown to be more sensitive than the Pap test for detection of cervical cancer. Currently, a variety of PCR assays detect other pathogens as well as pathogen resistance to specific drugs.
Although most of the initial PCR-based IVD tests were for infectious disease pathogens, many PCR assays now involve genetic targets, such as cystic fibrosis carrier screening, and many focus on detecting inherited (e.g., BRACA1 and BRACA2) mutations and somatic mutations in tumors (e.g., KRAS).
A new class of PCR-based assays reflects the emergence of personalized medicine: these assays identify subsets of patients who will benefit from a specific drug, either by avoiding an adverse effect or by heightened therapeutic response. In some cases, the assay is co-developed with the drug and is known as a companion diagnostic (e.g., Zelboraf, which targets melanoma tumors harboring the BRAF V600E mutation).
The early diagnostic assays required a manual DNA extraction step. The molecular-diagnostic goal, however, is a “sample in, clinical results out” platform. Today, many approved IVD platforms have coupled an automated DNA extraction method to an analytic method.
So, like the assays themselves, PCR diagnostic instruments have been getting “better”—better in the sense of bigger (for high sample throughput) or smaller (for point-of-care assays), but always faster, using rapid thermal cycling.
Some analytic methods, however, such as NGS are still relatively complex for complete automation and, for the moment, are offered as lab-developed tests in CLIA certified labs.
Almost 30 years after the first publication, PCR remains a critical tool in basic research and clinical application. For the foreseeable future, as new technologies are developed, PCR will likely continue to be an indispensable part of these and existing nucleic acid-based analyses.
Henry A. Erlich, Ph.D. ([email protected]), is director of human genetics and vp of discovery research at Roche Molecular Systems. His interests have focused on the development and application of new genotyping technologies and analytic methods to the study of disease, forensics, evolution, and population genetics. In the mid 1980s, Dr. Erlich, Kary Mullis, Ph.D., and their Cetus colleagues pioneered the development of PCR and its application to genetic diagnostics starting with sickle cell anemia and, in 1986, the first forensics application of PCR-based DNA testing.
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