Health Sciences Radiolabeld DNA

Question: Discuss about the Health Sciences for Radiolabeld DNA. Answer: Introduction Two research groups independently developed In situ hybridization (ISH). The 28S RNA or Radiolabeld DNA was hybridized to cytological preparations from the oocytes of Xenopus and was detected through microautoradiography. The examination of nucleic acid sequences inside the cells has been allowed by this technique and it had not altered the morphology or integrity of the cell and its different components. Since then, the modification of ISH has been done for studying the evolution of chromosomes, chromosomal analysis of leukaemias and tumors together with the cytogenetic studies of a large number of species. Drs. Nielsen, Egoholm, Buchardt and Berg carried out the discovery of Peptide Nucleic Acid (PNA) for the first time in the year 1991. PNA consists of a polyamide backbone i.e. the nucleobases that are modified and have extraordinary physical, biological and chemical properties like higher binding affinity, exceptional biological stability, blocking enzyme function, better specifi city, a probe for hybridization, cellular uptake, molecular diagnostics, labelling of plasmids with diverse kinds of fluorescent molecules and several other applications in the field of biomedical sciences. The potential applications of PNA include supramolecular nanostructure, antisense technology, nanoelectrical system, antisense technology and DNA computing. It also has nanomedical applications together with drug delivery and diagnostics for treating the microbial infections as well as diseases. In addition, the knowledge and understanding regarding the biological processes like protein synthesis and gene expression not only helps in the development of the procedures related to medical diagnostics, but are also useful in the medical treatment through the introduction of gene and antisense therapy (1). PNA has properties such as high sensitivity, high binding affinity and high specificities that have been explored in the PNA array that leads to the formation of a duplex of PNA/DNA because of the electrically neutral property of the oligomers of PNA. These strong duplexes of PNA/DNA bring about higher melting temperature (2). PNA probes possess high biological stability and are resistant to degradation by enzymes due to the presence of a backbone of N(2aminoethyl)glycine, which is not recognized by the proteases and nucleases. Since the enzymes cannot degrade the probes of PNA, the shelf life of of these probes is significantly long over years even at room temperature (3). Peptide Nucleic Acids have different applications based on its distinctive biophysical properties and have drawn the attention of molecular biologists, biochemists, chemists, material engineers, biotechnologists and material engineers for the development of genetic diagnostics, gene therapeutic drugs, identification of viral or bacterial contaminants in biological samples, as probes for FISH, microarray technology and DNA biosensors (4). The exceptional physicochemical properties of of PNA molecules, facilitates the development of assays that are uncomplicated and robust in several areas of biology together with molecular genetics, virology, microbiology, cytogenetics, and parasitology because of the chemical modifications that are new to the original backbone of PNA. This kind of modification may add to enhance PNAs potentialities for new applications in diagnostics and research like analysis of chromosome, analysis of point mutation, mycology, bacteriology, human pathology, and potential use in the form of therapeutic agents (5). The probes of PNA also provide an exceptional opportunity for specifically identifying microorganisms in the specimens of pathology, together with paraffin embedded and formalin fixed material. This particular technology associates pathology and clinical microbiology, provides a new format for testing, and opens the door to additional applications of test in clinical laboratory. The probe of PNA combines the simplicity with specificity and sensitivity of molecular technologies. This type of combination is compatible for replacing the current technologies, which provide fast and specific diagnosis and tests of microbiology that result in time of an appropriate therapy of a patient (6). The potential applications of PNA comprise supramolecular nanostructure, antisense technology, nanoelectrical system, antisense technology and DNA computing. It also has nanomedical applications together with delivery of drugs and diagnostics for treating the microbial infections as well as diseases. Additionally, the knowledge and understanding concerning the biological processes such as protein synthesis and gene expression not only assists in the development of the methods associated with medical diagnostics, but are also helpful in the medical treatment by the introduction of gene and antisense therapy. PNA has properties such as high sensitivity, high binding affinity and high specificities that have been explored in the PNA array that leads to the formation of a duplex of PNA/DNA because of the electrically neutral property of the oligomers of PNA. These strong duplexes of PNA/DNA bring about higher melting temperature. PNA probes possess high biological stability and are resis tant to degradation by enzymes due to the presence of a backbone of N(2aminoethyl)glycine, which is not recognized by the proteases and nucleases. Since the enzymes cannot degrade the probes of PNA, the shelf life of of these probes is considerably long over years. Robustness of PNA and its affecting factors The PNA probes are provided with the unique characteristics of hybridization like stronger and rapid binding to the complementary targets by the synthetic backbone. These properties of the molecules of PNA are elucidated by the lack of electrostatic repulsion characteristically encountered when the hybridization of negatively charged complementary oligomers of DNA occurs. Due to the presence of a non-charged backbone, the probes of DNA hybridize independent of the salt concentration. In addition, the physico-chemical behaviour of the probes of PNA comparative to DNA gets impacted due to the negative charges. They have facilitated the development of exceptional hybridization of PNA and PNA/target separation methods. All these methods encompass pre-gel hybridization as well as binding of hybrids of probe/target to the surfaces and polymers that are positively charged (7). The unnatural backbone of PNA also signifies that the degradation of PNA do not occur by the ubiquitous enzymes like proteases and nucleases. The elevated biostability is not only essential for their utilization as therapeutic antisense agents, but is also probable to enhance the stability of probes of PNA in the diagnostic applications and offer enhanced shelf-life of the product together with a better range of assay formats. Due the backbone, the recognition of PNA do not take place by polymerases and hence cannot be used directed or copied as a primer and the monomers of PNA cannot be incorporated enzymatically into amplicons. Characteristics make the self-reporting probe of PNA more robust molecules for detection involving the real-time PCR methods as compared to their counterparts that are derived from DNA, for example the hybridization probes for the light cycler that are degraded throughout PCR by means of Taq DNAs endonuclease activity (8). In reality, the variables like kind of fixative utilized (alcohol-based or aldehyde fixation) temperature, hybridization time, probe concentration, pH, formamide and dextran sulphate, among others, are identified to affect the efficiency of hybridization (9). The time and temperature of hybridization are important variables for the outcome of the process of hybridization and for lowering the temperature at which hybridization will be performed; formamide is most commonly used (10). The temperature of hybridization is associated with the affinity of the probe to the target and its estimation can be done by Gibbs free energy change, which is related to the reaction of hybridization. The time of hybridization has been linked with the process kinetics that encompasses probe penetration through the envelope of the cell, the probe binding with the complementary sequence together with the unfolding of secondary as well as tertiary structures of rRNA and probes eventual folded portions (11). For reducing the thermal stability of the double-stranded polynucleotides, Formamide (FA), which is a denaturing agent, is used. It enhances the accessibility of the target of rRNA and competes for hydrogen bonding that facilitates the hybridization to be performed at the lower temperatures. Hence, it has been presumed that the concentration of of FA required, together with the time and temperature of hybridization. It regulates the stringency of the process and is dependent only on the sequence of the target and the structure of the probe. Additionally, the physico-chemical behaviour of the probes of PNA comparative to DNA gets impacted due to the negative charges (12). For lowering the annealing temperature and boiling point of the strands of the nucleic acids in In situ hybridization (ISH), formamide is an ideal solvent. It has an advantage for preserving the morphology because of a lower temperature for incubation. Nonetheless, in fluorescence in situ hybridization (FISH), aligned with the unique targets of DNA in tissue sections, for obtaining the adequate signal testing, an overnight hybridization is needed. Over the past 30 years, for in situ hybridization, the solvent for choice is formamide. It lowers the melting point through the destabilization of the double-stranded structure of the nucleic acid helix. Its toxicity is distinguished but has been overshadowed by its advantageous effects. When the hybridizing DNA probes to low copy number or single locus targets on the sections of Formalin-fixed, paraffin-embedded tissue, an incubation of 16 hours or more is needed and is the major time consuming step in the procedure of in situ hybridizatio n. If the hybridization of the entire genome is done, for instance, with comparative genomic hybridization, a hybridization time 49 to 94 hours is often utilized. A major disadvantage of Fish utilizing the probes of oliginucleotides is the inconsistent and at times inadequate penetration of probes in the bacteria depending on the characteristics of their cell wall. In acid-fast bacilli and Gram-positive species, this is observed mainly as a problem. PNAs may be helpful to overcoming this problem. Because of the presence of their neutral backbone, the diffusion of PNAs occurs through the hydrophobic cell walls and allows the detection of mycobacterium through FISH and do not involve any pre-treatment. Utilising the specific fluorescently labelled PNAs, the differentiation between non-tuberculous and tuberculous mycobacteria was possible in the smears of mycobacterial cultures and directly in the samples of smear-positive sputum within few hours. However, in fluorescence in situ hybridization (FISH), aligned with the distinctive targets of DNA in tissue sections, for obtaining the sufficient signal testing, an overnight hybridization is required . This method constitutes an enhancement in the regular diagnosis of tuberculosis and may assist in establishing FISH as a fast, cost-effective and valuable method in the field of clinical microbiology. An organism concentration of no less than 105CFU/ml is required by PNA-FISH for the rocess of detection. This is a limitation of PNA-FISH and this particular requirement may prove to be difficult for detecting the fastidious or slow-growing organisms. Generally, the probes of DNA shorter in comparison to the conventional nucleotides are needed for the specific binding. Therefore, they appear to be an interesting substitute to the conventional oligonucleotide probes. In comparison to aegPNA and DNA, it presented a better sequence specificity and a strong binding affinity towards DNA. Method available for detecting MRNA in the tissue section An essential tool for studying the spatial organization of the genome with a high accuracy is Fluorescent in situ hybridization (FISH). The labelled probes that target the entire chromosome or regions of chromosomes allow the direct visualization in the interphase as well as the metaphase (6). The generation of such painted probes can be done from the DNA isolated by the microdissection of the metaphase chromosome, followed by the amplification and labelling with the nucleotides that are modified by degenerate oligonucleotide primer- PCR(DOP-PCR). This method comprises the step of universal amplification that is mainly effective at amplifying the single copies of chromosome to produce paints or perform additional cytogenetic applications in which there is an availability of small quantity of DNA, for instance small pieces of microdissected tissue or single cells (7). On the other hand, Chromogenic in situ hybridization (CISH), is an appropriate substitute to FISH. It leads to the pro duction of a permanent chromosome by utilising peroxidase or alkaline phosphatase- labelled reporter antibodies, which interacts with the probes of the hybridized DNA that are subsequently detected by means of an enzymatic reaction (8). CISH is advantageous over FISH as it can be viewed through a bright-field microscope. For the rapid diagnosis of bacterial pathogens in the microbiology laboratory, Real-time (RT) PCR testing can be reliable as it is an emerging competing technology. It is probable to challenge directly PNA FISH (13). Unlike PNA FISH that assists in the detection of RNA in the living organisms and it can also detect the infections that are ongoing in nature, RT-PCR cannot distinguish between the past and current infection, and therefore it is very sensitive for the diagnosis, but may possibly lack clinical specificity (14). Its development was carried out for rapidly enhancing the process of DNA amplification and improvement of specificity and sensitivity over the traditional methods. The testing can be done on all the types of specimen, along with paraffin specimens, and combines DNA technology with the fluorescent probes of the product, which is amplified in the similar reaction vessel. Since the reaction takes place within a closed vessel, the risk of contamination of the environme nt is reduced and needs less time and produce rapid results within an hour in comparison to the traditional methods of PCR testing (15). A better healthcare can be achieved by rapid and accurate diagnosis and PNA probe is more efficient than the traditional methods in this context. In the present scenario, the rate at which the human pathogens are getting resistant is alarming and it is leading towards an urgent need for improving the diagnostic technologies that are intended for the rapid detection along with point-of-care testing for supporting the quick decision making concerning the management of patient and antibiotic therapy. These data reveal the advantage of rapid that can reduce the treatment of the cultures that are contaminated, reduce expenses and stay in the hospital and it may decrease antimicrobial resistance. A disparity among the empirical therapeutic and ensuing susceptibility result from a specific organism is one of the important factor that leads to the delaying of an effectual therapy (16). It has been revealed that early and rapid administration of antimicrobial therapy to the patients suffering from the infection of the bloodstream results in the reduction of mortality. In addition, the need for a rapid diagnosis has usually been overlooked due the practices of empiric and prophylactic treatment, which utilizes broad-spectrum antibiotics. At present, though, it is well established that more suitable utilization of antibiotics is needed for limiting the emergence of the pathogens that are resistant to multiple drugs. Altering the empiric nature of antibiotics, therapy in which the patients are covered with antibiotics of a broad spectrum needs new, fast and perfect tools of diagnosis for providing the physician with timely and reliable information for the therapy and management of patients (17). Clinical microbiology laboratory service is intended for detecting and identifying the specific microorganisms in the clinical specimens for diagnosing the infectious diseases. Nonetheless, the significance of precision and the ease of the slow, inexpensive methods frequently delay the reporting time for a number of days or weeks (18). The exceptional performance distinctiveness of PNA FISH are attributed to the high specificity of the probes of PNA integrated with the utilization of rRNA as a target and they evidently demonstrate how molecular diagnosis can offer accurate results within a particular frame of time and is not likely to use the traditional phenotype-based methods of identification. In addition, PNA FISH is better than the traditional methods for mixed cultures like Staphylococcus aureus bacteremia contaminated with coagulase-negative Staphylococcus species (CNS) and for almost the species that are indistinguishable, for example C.dubliniensis and C. Albicans, in which the traditional methods failed to, provide an accurate diagnosis (6). Today, diagnostic testing is developing to become a significant constituent of the modern medicine, which is based on information and focuses on to provide rapid as well as accurate results potentially resulting in the better treatment of patients, controlling infection, and management of healthcare. PNA FISH is an illustration of diagnosis tests in this new generation and is intended to provide a real-time impact on the therapy of the patients (18). Through the introduction of PNA FISH for the rapid diagnosis of the infections in the bloodstream, novel tools has been provided to the clinical microbiology laboratory for reporting the diagnostic results in time to select the correct therapy. This evidently exemplifies how the first therapy-directing diagnosis will target the diagnostic needs that are not met and are related to the current practices of treatment with the antibiotics that are commonly used (6) In the end, it can be concluded that precise diagnostic tests have a key role in the management of patient and the control of most irresistible illnesses. Unfortunately, in numerous developing countries, clinical care is frequently compromised by the absence of regulatory controls on the quality of these tests. The data accessible on the execution of a diagnostic test can be one-sided or defective as a result of failings in the configuration of the studies which evaluated the performance attributes of the test. Subsequently, diagnostic tests are sold and utilized in much of the developing world without confirmation of adequacy. Misdiagnosis leading to failure in treating a severe disease or squandering costly treatment on individuals who are not infected remains a serious problem of health and wellbeing. The improvement of a diagnostic test more often than not takes after a way from recognizable proof of the diagnostic target and advancement of test reagents to the improvement of a t est model. The exceptional physicochemical properties of of PNA molecules, facilitates the development of assays that are uncomplicated and robust in several areas of biology together with molecular genetics, virology, microbiology, cytogenetics, and parasitology because of the chemical modifications that are new to the original backbone of PNA. Currently, the rate at which the pathogens of humans are getting resistant is frightening and it is leading towards an urgent need for enhancing the diagnostic technologies that aimed to the rapid detection along with point-of-care testing for supporting the quick decision making concerning the patient management and antibiotic therapy. It shows the decrease in the treatment of the cultures that are contaminated, lessen expenses and stay in the hospital and it may reduce antimicrobial resistance. A difference among the empirical therapeutic and subsequent susceptibility result from a specific organism is one of the significant factor that le ads to the delaying of an efficient therapy.