Molecular strategies for the study of the expression of gene variation by real-time PCR

• Published in: The Handbook of Microbial Bioresources pp. 591 - 615
• Main Author: Cristóbal, H. A
• Format: Book Chapter
• Language: English
• Published: Wallingford, UK CABI 2016; CAB International
• Subjects: Alteromonadales; Bacteria; bacterium; biochemical genetics; Biotechnology; Botany & plant sciences; cultures; deoxyribonucleic acid; DNA; DNA sequences; Gammaproteobacteria; gene expression; genes; genetic variability; genetic variation; genotypic variability; genotypic variation; mathematical models; messenger RNA; Microbiology (non-medical); molecular genetics; molecular genetics techniques; mRNA; nucleotide sequences; PCR; polymerase chain reaction; prokaryotes; Proteobacteria; real time PCR; ribonucleic acid; ribosomal DNA; RNA; Shewanella; Shewanellaceae
• ISBN: 9781780645216; 178064521X
• DOI: 10.1079/9781780645216.0591

All living organisms regulate their activities through the activation or repression of expression of their genes. The genetic expression of a target gene is generally proportional to the number of copies of messenger RNA (mRNA). Therefore, for detecting specific cell products it is crucial to identify the number of copies of mRNA when it is translated to form proteins. This measure allows obtaining data on the production of biological elements and the varying levels of expression of its genes in the cell in response to exposure to various effects. The identification of specific sequences (DNA or RNA) and detection of the level of genetic expression represents important data in several areas such as medicine, biotechnology and the food industry. A number of diverse molecular biology techniques are capable of detecting genetic expression, for example northern blotting, microarrays, capillary electrophoresis and real-time PCR. Real-time PCR has become one of the most widely employed methods for gene quantification. This technique provides a high data output with a high level of sensitivity that is able to detect specific sequences; it does not require post-amplification manipulation and a large number of samples can be processed using modern equipment that avoids laboratory contamination. Nevertheless, several considerations must be taken into account when working with real-time PCR, such as the choice of the strategy of quantification, the choice of fluorescent markers and how the data is to be interpreted. This chapter covers the basic concepts of the real-time PCR technique and handling the data obtained, including types of absolute and relative quantification, mathematical models available for relative quantification and amplification efficiency calculations, types of normalization, chemicals used and applications in different fields of research. In addition, various steps for starting real-time PCR in order to determine gene expression levels of 16SrDNA, , , and from sp. G5 cultures are evaluated and optimized.