Application of Molecular Bio Techniques Lab - Nathan...

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Nathan Heckendorf BIO-181L 11/16/16 Professor Hite Application of Molecular Biology Techniques Lab Introduction The development of molecular biology was one of the greatest achievements in biological science in the century XX. The discovery of Polymerase Chain Reaction (PCR) brought enormous benefits and scientific developments such as genome sequencing, gene expressions in recombinant systems, the study of molecular genetic analyses, including the rapid determination of both paternity and the diagnosis of infectious diseases (Reece et al., 2014). PCR enables the in vitro synthesis of nucleic acids through which a DNA segment can be specifically replicated in a semi-conservative way. It generally exhibits excellent detection limits. Recent developments in molecular methods have revolutionized the detection and characterization of microorganisms in a broad range of medical diagnostic fields, including virology, mycology, parasitology, microbiology and dentistry. Among these methods, Polymerase Chain Reaction (PCR) has generated great benefits and allowed scientific advancements. PCR is an excellent technique for the rapid detection of pathogens, including those difficult to culture (Areda, Boyles, Francis, & Hite, 2016). Along with conventional PCR techniques, Real-Time PCR has emerged as a technological innovation and is playing an ever-increasing role in clinical diagnostics and research laboratories. Due to its capacity to generate both qualitative and quantitative results, Real-Time PCR is considered a fast and accurate platform. The possibility of Real-Time PCR monitoring has revolutionized the quantification process of DNA and RNA fragments. Real- Time PCR allows the precise quantification of these nucleic acids with greater reproducibility. Real-Time PCR requires a thermocycler with an optical system to capture fluorescence and a computer with software capable of capturing the data and performing the final analysis of the reaction. The programs available from diverse manufactures exhibit differences regarding sample capacity, method of excitation and total sensitivity. There are also differences between regarding the data processing. The emission of fluorescence generates a signal that increases in direct proportion with the amount of PCR products (Reece et al., 2014). Fluorescence values are recorded during each cycle and represent the amount of amplified product. Conventional PCR has been used for over a decade in clinical microbiology laboratory research for the identification of microbial pathogens. However, for a number of reasons, this technique has been restricted to the detection of microorganisms that either have slow growth or cannot be cultivated. Most tests based on conventional PCR involve multiple steps and, therefore, require careful expertise.

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