In biochemistry, denaturation is a process in which proteins or nucleic acids lose the quaternary structure, tertiary structure, and secondary structure which is present in their native state, by application of some external stress or compound such as a strong acid or base, a concentrated inorganic salt, an organic solvent, agitation and radiation or heat. If proteins in a living cell are denatured, this results in disruption of cell activity and possibly cell death. Protein denaturation is also a consequence of cell death. Denatured proteins can exhibit a wide range of characteristics, from conformational change and loss of solubility to aggregation due to the exposure of hydrophobic groups. The loss of solubility as a result of denaturation is called coagulation. Denatured proteins lose their 3D structure and therefore cannot function.
Gel electrophoresis is a method for separation and analysis of biomacromolecules and their fragments, based on their size and charge. It is used in clinical chemistry to separate proteins by charge or size and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge.
A chaotropic agent is a molecule in water solution that can disrupt the hydrogen bonding network between water molecules. This has an effect on the stability of the native state of other molecules in the solution, mainly macromolecules by weakening the hydrophobic effect. For example, a chaotropic agent reduces the amount of order in the structure of a protein formed by water molecules, both in the bulk and the hydration shells around hydrophobic amino acids, and may cause its denaturation.
The first isolation of deoxyribonucleic acid (DNA) was done in 1869 by Friedrich Miescher. DNA extraction is the process of isolating DNA from the cells of an organism isolated from a sample, typically a biological sample such as blood, saliva, or tissue. It involves breaking open the cells, removing proteins and other contaminants, and purifying the DNA so that it is free of other cellular components. The purified DNA can then be used for downstream applications such as PCR, sequencing, or cloning. Currently, it is a routine procedure in molecular biology or forensic analyses.
Liquid–liquid extraction (LLE), also known as solvent extraction and partitioning, is a method to separate compounds or metal complexes, based on their relative solubilities in two different immiscible liquids, usually water (polar) and an organic solvent (non-polar). There is a net transfer of one or more species from one liquid into another liquid phase, generally from aqueous to organic. The transfer is driven by chemical potential, i.e. once the transfer is complete, the overall system of chemical components that make up the solutes and the solvents are in a more stable configuration. The solvent that is enriched in solute(s) is called extract. The feed solution that is depleted in solute(s) is called the raffinate. LLE is a basic technique in chemical laboratories, where it is performed using a variety of apparatus, from separatory funnels to countercurrent distribution equipment called as mixer settlers. This type of process is commonly performed after a chemical reaction as part of the work-up, often including an acidic work-up.
Aqueous biphasic systems (ABS) or aqueous two-phase systems (ATPS) are clean alternatives for traditional organic-water solvent extraction systems.
Guanidinium thiocyanate(GTC) or guanidinium isothiocyanate (GITC) is a chemical compound used as a general protein denaturant, being a chaotropic agent, although it is most commonly used as a nucleic acid protector in the extraction of DNA and RNA from cells.
A plasmid preparation is a method of DNA extraction and purification for plasmid DNA, it is an important step in many molecular biology experiments and is essential for the successful use of plasmids in research and biotechnology. Many methods have been developed to purify plasmid DNA from bacteria. During the purification procedure, the plasmid DNA is often separated from contaminating proteins and genomic DNA.
Ethanol precipitation is a method used to purify and/or concentrate RNA, DNA, and polysaccharides such as pectin and xyloglucan from aqueous solutions by adding ethanol as an antisolvent.
Isoamyl alcohol is a colorless liquid with the formula C
5H
12O, specifically (H3C–)2CH–CH2–CH2–OH. It is one of several isomers of amyl alcohol (pentanol). It is also known as isopentyl alcohol, isopentanol, or (in the IUPAC recommended nomenclature) 3-methyl-butan-1-ol. An obsolete name for it was isobutyl carbinol.
Acid guanidinium thiocyanate-phenol-chloroform extraction is a liquid–liquid extraction technique in biochemistry. It is widely used in molecular biology for isolating RNA. This method may take longer than a column-based system such as the silica-based purification, but has higher purity and the advantage of high recovery of RNA: an RNA column is typically unsuitable for purification of short RNA species, such as siRNA, miRNA, gRNA and tRNA.
DNA separation by silica adsorption is a method of DNA separation that is based on DNA molecules binding to silica surfaces in the presence of certain salts and under certain pH conditions.
In molecular biology, quantitation of nucleic acids is commonly performed to determine the average concentrations of DNA or RNA present in a mixture, as well as their purity. Reactions that use nucleic acids often require particular amounts and purity for optimum performance. To date, there are two main approaches used by scientists to quantitate, or establish the concentration, of nucleic acids in a solution. These are spectrophotometric quantification and UV fluorescence tagging in presence of a DNA dye.
Acid–base extraction is a subclass of liquid–liquid extractions and involves the separation of chemical species from other acidic or basic compounds. It is typically performed during the work-up step following a chemical synthesis to purify crude compounds and results in the product being largely free of acidic or basic impurities. A separatory funnel is commonly used to perform an acid-base extraction.
TRIzol is a widely used chemical solution used in the extraction of DNA, RNA, and proteins from cells. The solution was initially used and published by Piotr Chomczyński and Nicoletta Sacchi in 1987.
RNA extraction is the purification of RNA from biological samples. This procedure is complicated by the ubiquitous presence of ribonuclease enzymes in cells and tissues, which can rapidly degrade RNA. Several methods are used in molecular biology to isolate RNA from samples, the most common of these is guanidinium thiocyanate-phenol-chloroform extraction. The filter paper based lysis and elution method features high throughput capacity.
Spin column-based nucleic acid purification is a solid phase extraction method to quickly purify nucleic acids. This method relies on the fact that nucleic acid will bind to the solid phase of silica under certain conditions.
Boom method is a solid phase extraction method for isolating nucleic acid from a biological sample. This method is characterized by "absorbing the nucleic acids (NA) to the silica beads".
Phenol–chloroform extraction is a liquid-liquid extraction technique in molecular biology used to separate nucleic acids from proteins and lipids.
Small RNA sequencing is a type of RNA sequencing based on the use of NGS technologies that allows to isolate and get information about noncoding RNA molecules in order to evaluate and discover new forms of small RNA and to predict their possible functions. By using this technique, it is possible to discriminate small RNAs from the larger RNA family to better understand their functions in the cell and in gene expression. Small RNA-Seq can analyze thousands of small RNA molecules with a high throughput and specificity. The greatest advantage of using RNA-seq is represented by the possibility of generating libraries of RNA fragments starting from the whole RNA content of a cell.
