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Apr 1, 2013 (Vol. 33, No. 7)

Development and Evolution of PCR

  • Quantification

    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.

  • Applications

    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).

  • Personalized Medicine

    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.

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