PCR – diagnostics. Aizhan Zhussupova
DNA are obtained. It permits to estimate the presence of a pathogen in a sample, even if there are only a few DNA molecules of the pathogen. PCR may prove to be very useful in the diagnosis of chronic, persistent infections, such as bovine viral diarrhoea, enzootic bovine leukosis or caprine arthritis/encephalitis virus. These diseases present serious problems in terms of diagnosis and prevention as infected animals are a constant potential source of transmission. It also allows to diagnose the presence of slowly growing pathogens, without resorting to timeconsuming microbiological methods, which is especially important in gynecology and urology in the diagnosis of urogenital infections and sexually transmitted diseases.
Also, this method diagnoses viral infections, such as hepatitis, human immune-deficiency virus, and others. The sensitivity of the method is higher than that of immune chemical and microbiological methods, and the principle of the method allows diagnosing the presence of infections even with significant antigenic variation.
Specificity of PCR – diagnostics for a broad range of viral, chlamydia, mycoplasma, ureaplasma, and large number of other bacterial infections reaches 100 %. PCR – diagnostics allows detection of infectious agents even in cases, where other methods (immunological, bacteriological, and microscopic) cannot do so.
PCR – diagnostics is particularly effective for diagnosis of hardly cultured, uncultured and private existing forms of microorganisms, which often are encountered in the latent and chronic infections, permitting to avoid the difficulties associated with the cultivation of microorganisms in the laboratory. Use of PCR diagnostics is also very effective against pathogens with high antigenic variability and intracellular parasites.
By the PCR means detection of pathogens is possible not only in clinical material obtained from the patient, but also in materials derived from the environmental objects (water, soil, etc.): in urological and gynecological practice – for the detection of chlamydia, ureaplasma, gonorrhea, herpes, bacterial vaginosis, mycoplasma infection, human papilloma virus; in pulmonology – for the differential diagnosis of viral and bacterial pneumonia, tuberculosis; in gastroenterology – in order to identify helicobacteriosis; in clinics of infectious diseases – as a rapid method of diagnosis of salmonellosis, diphtheria, hepatitis B, C, and G; in hematology – in order to detect cytomegalovirus infection, oncoviruses.
The technique is also used for rapid prenatal diagnosis and carrier testing of several inherited disorders. Prospective parents can be tested for being genetic carriers, or their children might be tested for actually being affected by a disease. After PCR, mutations producing singlegene disorders can be detected by several different methods, including endonuclease digestion, gel electrophoresis, and hybridization to an oligonucleotide probe specific for a mutation. Less often, gene sequencing of a PCR product is used to rapidly identify a mutation. In addition, the PCR technique can be applied to polymorphism analysis to provide diagnosis by linkage analysis.
A new generation of robotic workstations is now available where PCR reactions may be set up with only a single tube open at any one time. This greatly reduces the risk of contamination. It is also important to control for potential ‘negative’ results caused by the presence of PCR inhibitors in the reaction mixture. A template, independent of the target DNA, known to produce a PCR product (mimics) with specific primers can be used as a control for the PCR inhibitors, thus indicating falsenegative results. Use of these precautions allows the PCR to become a realistic option for the diagnostician. Currently, PCR has been applied to many areas of research in molecular genetics: generating hybridization probes for Southern or northern blot hybridization, numerous applications to DNA cloning, sequence-tagged sites, phylogenic analysis of DNA from ancient sources, study of patterns of gene expression, genetic mapping by studying chromosomal crossovers after meiosis.
PCR analysis is also essential to preimplantation genetic diagnosis, where individual cells of a developing embryo are tested for mutations. PCR can also be used as part of a sensitive test for tissue typing, vital to organ transplantation; study of cancer associated mutations facilitates therapy regimens to be individually customized to a patient; DNA fingerprinting is used in parental testing and forensics.
Manual comprises basic theoretical questions of modern PCR – diagnostics, including its components and stages, its detection and analysis, primer and probes design, and its practical application in the field of molecular biology, genetic engineering and medicine, and in the field of laboratory diagnostics of hereditary and infectious diseases in particular, control questions and sample tests; is well illustrated with schemes and figures.
It might be recommended as additional literature for specific and general courses in the field of biology and biotechnology and primarily for such courses as «Molecular biology», «General and molecular genetics», «Problems in modern biology». Manual might also be interesting for a broader group of readers.
Chapter 1
INVENTION OF THE POLYMERASE CHAIN REACTION
«My little silver Honda s front tires pulled us through the mountains. My hands felt the road and the turns. My mind drifted back into the laboratory. DNA chains coiled and floated. Lurid blue and pink images of electric molecules injected themselves somewhere between the mountain road and my eyes. I see the lights on the trees, but most of me is watching something else unfolding… I didn’t sleep that night. The next morning I bought two bottles of Navarro Vineyards Pinot Noir, and by mid afternoon had settled into a fitful sleep. There were diagrams of PCR reactions on every surface that would take pencil or crayon in my cabin. I woke up in a new world».
Before PCR, molecular biologists utilized nucleic acid sequence data (sequence motifs) to design «hybridization» probes for use in assays for the detection and identification of specific RNA and DNA fragments (moieties). These «hybridization» assays were used to determine the presence/absence of specific RNA or DNA sequences within complex mixtures of nucleic acids, via the use of specifically designed (semisynthetic) complementary DNA or RNA molecules (nucleic acid probes) which had been equipped with radioactive labels for detection purposes. The target nucleic acid population was initially attached to a solid carrier phase and then hybridized with specific labeled probe. After stringent hybridization and extensive washing procedures, the presence of the target DNA fragment could be determined by the presence/absence of the radioactively labeled probe on solid carrier phase.
Alternatively, direct visualization of the probe and target molecule was achieved via electron microscopy, with hybridization being quantified on the basis of the different widths of double stranded (hybridized) versus single stranded (non-hybridized) nucleic acids. The most convenient of these hybridization test systems utilized filter hybridization (where the target DNA extract was first immobilized on nitrocellulose or nylon filters), in combination with post-hybridization autoradiography or scintillography. This format greatly increased test sensitivity and vastly improved the technical reliability and speed of the hybridization procedure, allowing the detection of picogram quantities of target material. However, one major disadvantage of these hybridization systems was the requirement for radioactively labeled probes, not least because working with radioactive materials is hazardous for your health, requires legal permits, correct disposal systems, and is relatively expensive to use. For these reasons, radioactive labels have been largely replaced by various (non-radioactive) chemical labels, facilitating the development of colorimetric, chemoluminescent and chemo-fluorescent hybridization detection methods. However, these «second generation)) chemical-labeling and detection systems do not generally yield as high a degree of sensitivity as the original radio-labeling and detection systems, though both systems are amenable to automation and high throughput applications. To date, a variety of elegant techniques based upon the basic hybridization principle have been developed (e.g. sandwich hybridization, Southern- and Northern-blot hybridizations, etc.) and these are frequently applied in both fundamental research and clinical diagnostics. The need to detect very small numbers of clinically relevant molecules was high. For instance, the detection of low-titer viral infections, minimal residual disease in leukemia patients, point mutations in genes or genetic aberrations in tumors etc., all require highly sensitive methodologies. This has led to the development of novel approaches specifically aimed at the amplification of target (gene) sequences prior to detection, such that sensitivity issues related to hybridization/probe detection protocols would no longer be the limiting step of DNA and RNA