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Cloning of genes for protein expression
Published in Raimund J. Ober, E. Sally Ward, Jerry Chao, Quantitative Bioimaging, 2020
Raimund J. Ober, E. Sally Ward, Jerry Chao
In the following sections, we describe how molecular biology tools can be used to generate expression plasmids that can be used, in the context of this book, for the production of antibodies, proteins fused to fluorescent proteins, or modified proteins that can be site-specifically labeled with fluorophores. These sections are included for completeness, but are not essential for understanding the general principles. Before describing the detailed methods, we first discuss two important tools that are used in molecular biology, namely restriction enzymes and the polymerase chain reaction.
Genes and Genomics
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2020
Nucleases are enzymes that cut DNA strands by catalyzing the hydrolysis of the phosphodiester bonds. Nucleases that hydrolyze nucleotides from the ends of DNA strands are called exonucleases, whereas endonucleases cut within strands. The most frequently used nucleases in molecular biology are the restriction endonucleases, which cut DNA at specific sequences. For instance, the EcoRV enzyme recognizes the 6-base sequence 5′-GAT|ATC-3′ and makes a cut at the vertical line. In nature, these enzymes protect bacteria against phage infection by digesting the phage DNA when it enters the bacterial cell, acting as part of the restriction modification system. In technology, these sequence-specific nucleases are used in molecular cloning and DNA fingerprinting. Enzymes called DNA ligases can rejoin cut or broken DNA strands. Ligases are particularly important in lagging strand DNA replication, as they join together the short segments of DNA produced at the replication fork into a complete copy of the DNA template. They are also used in DNA repair and genetic recombination.
Innovations in Noninvasive Instrumentation and Measurements
Published in Robert B. Northrop, Non-Invasive Instrumentation and Measurement in Medical Diagnosis, 2017
The polymerase chain reaction (PCR) is a molecular biology/biochemical protocol used to exactly replicate (amplify) a single piece of DNA (oligo) from a sample to create thousands to millions of copies of that particular DNA sequence (Riley 2005). These copies allow other analytical techniques to more easily identify the base sequences of the oligo. The PCR allows the DNA from a sample as small as a fraction of a genome, a single-cell nucleus, or a mitochondrion to be characterized in terms of its base sequences. The PCR has many applications, ranging from criminal science to genomics. However, in this section, we will address the PCR applications in medical diagnosis.
Development of thermally stable coarse water-in-oil emulsions as potential DNA bioreactors
Published in Journal of Dispersion Science and Technology, 2021
Maria Romero-Peña, Enders Kaion Ng, Supratim Ghosh
Polymerase chain reaction (PCR) is extensively utilized in molecular biology and applied microbiology to create copies of a specific deoxyribonucleic acid (DNA) segments and subsequent detection of a gene sequence.[1] In PCR, multiple thermal cycles are used for denaturation, hybridization, and polymerase extension of DNA segments from a small amount to a factor of 106.[2] Conventional single-molecule PCR amplifies several DNA molecules in a vial simultaneously to replicate and increase the DNA percentage.[3] Because the DNA amplification is done in a continuous aqueous phase, DNA quantification becomes extremely difficult at extremely low detection levels.[4]
Bacterial community in commercial airliner cabins in China
Published in International Journal of Environmental Health Research, 2020
Mingxin Liu, Junjie Liu, Jianlin Ren, Lumeng Liu, Ruiqing Chen, Yanju Li
Polymerase chain reaction technology (PCR) is a molecular biology technique used to amplify specific DNA fragments. It can be regarded as special DNA replication in vitro. The most prominent feature of PCR is that it can greatly increase trace amounts of DNA. PCR is the use of DNA denaturation, which will become single-stranded at high temperature (95°C) in vitro. When the temperature is low (usually around 60°C), the primers are combined with single-stranded base pairing. When adjusting the temperature to the DNA polymerase optimal reaction temperature (about 72°C), DNA polymerase along the direction of phosphoric acid to five carbon sugar synthesizes a complementary strand. With the characteristics of strong specificity, high sensitivity, simplicity, speed, and low purity requirements, PCR technology has been widely used in 16s rRNA identification.