Tell about the basic principles of digital techniques

#Tell about the basic principles of digital techniques software#

SYBR Green I is the most used dsDNA binding-specific dye in real-time PCR. The fluorescent composites used are SYBR В® Green and TaqMan В® ( 50, 73). Fluorescence values are recorded during each cycle and represent the amount of amplified product. The emission of fluorescence generates a signal that increases in direct proportion with the amount of PCR products. There are also differences between regarding the data processing. The programs available from diverse manufactures exhibit differences regarding sample capacity, method of excitation and total sensitivity.

#Tell about the basic principles of digital techniques software#

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. Advantages of Real-Time PCR include the ease of quantification, greater sensitivity, reproducibility and precision, rapid analysis, better control of quality in the process and a lower risk of contamination ( 62, 73). This technique provides a sensitive method for the accurate quantification of individual species, which could be very relevant to the diagnosis of pathogens and genetic diseases. Real-Time PCR allows the precise quantification of these nucleic acids with greater reproducibility. The possibility of Real-Time PCR monitoring has revolutionized the quantification process of DNA and RNA fragments.

Another useful PCR application is the cloning of a particular DNA fragment, which allows the study of gene expression and has considerable potential in forensic medicine ( 94). The development of tools for amplifying DNA segments has generated enormous benefits in gene analysis as well as the diagnosis of many genetic diseases and the detection of bacterial, viral and fungal pathogens ( 4, 72, 73, 99). Primers limit the sequence to be replicated and the result is the amplification of a particular DNA sequence with billions of copies ( 66, 73). This enzyme synthesizes a complementary sequence of DNA, as a small fragment (primer) is connected to one of the DNA strands in the specific site chosen to start the synthesis. PCR enables the synthesis of specific DNA fragments using a DNA-polymerase enzyme, which takes part in the replication of the cellular genetic material. Since its description, this technology has caused a veritable revolution in biological research, establishing the agreement of basic biological processes in applied areas involving diagnoses and genetic improvements for plants and animal ( 101). PCR was developed in the 1980s by Kary Mullis, who received the Nobel Prize in 1994 ( 14). It also seeks to evaluate and discuss the indications, uses and advantages of these techniques, as well as their advances in various medical areas. The aim of the present study is to outline the principles and applications of conventional PCR and Real-Time PCR techniques in some medical sciences.

This technique allows accompanying the reaction and presentation of results in a faster and more accurate fashion than conventional PCR, which only displays the qualitative results ( 50, 62, 73). Recently, a technological innovation of PCR, known as Real-Time PCR, has become increasingly important in clinical diagnostics and research laboratories due to its capacity for generating quantitative results. It generally exhibits excellent detection limits ( 19, 101). PCR enables the in vitro synthesis of nucleic acids through which a DNA segment can be specifically replicated in a semi-conservative way.

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 disease ( 73, 99). The development of molecular biology was one of the greatest achievements in biological science in the century XX.