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In biochemistry, denaturation is a process in which proteins or nucleic acids lose folded structure present in their native state due to various factors, including application of some external stress or compound, such as a strong acid or base, a concentrated inorganic salt, an organic solvent (e.g., alcohol or chloroform), agitation and radiation, or heat. [3]
Taq polymerase is a magnesium-dependent enzyme and determining the optimum concentration to use is critical to the success of the PCR reaction. [6] Some of the components of the reaction mixture such as template concentration, dNTPs and the presence of chelating agents ( EDTA ) or proteins can reduce the amount of free magnesium present thus ...
Exonuclease III (ExoIII) is an enzyme that belongs to the exonuclease family. ExoIII catalyzes the stepwise removal of mononucleotides from 3´-hydroxyl termini of double-stranded DNA . [ 1 ] A limited number of nucleotides are removed during each binding event, resulting in coordinated progressive deletions within the population of DNA molecules .
In the less extensive technique of equilibrium unfolding, the fractions of folded and unfolded molecules (denoted as and , respectively) are measured as the solution conditions are gradually changed from those favoring the native state to those favoring the unfolded state, e.g., by adding a denaturant such as guanidinium hydrochloride or urea.
A ten fold increase of monovalent cation concentration increases the temperature by 16.6 °C. The hyperchromic effect is the striking increase in absorbance of DNA upon denaturation. The two strands of DNA are bound together mainly by the stacking interactions, hydrogen bonds and hydrophobic effect between the complementary bases. The hydrogen ...
Guanidinium thiocyanate can be used to deactivate a virus, such as the influenza virus that caused the 1918 "Spanish flu", so that it can be studied safely.. Guanidinium thiocyanate is also used to lyse cells and virus particles in RNA and DNA extractions, where its function, in addition to its lysing action, is to prevent activity of RNase enzymes and DNase enzymes by denaturing them.
A chaotrope denatures biomolecules by disrupting the shell of hydration around them. This allows positively charged ions to form a salt bridge between the negatively charged silica and the negatively charged DNA backbone in high salt concentration. The DNA can then be washed with high salt and ethanol, and ultimately eluted with low salt.
Presence of certain ions in the reaction vessel also affects specific activity of the enzyme. Small amounts of potassium chloride (KCl) and magnesium ion (Mg 2+) promote Taq's enzymatic activity. Taq polymerase is maximally activated at 50mM KCl, while optimal Mg 2+ concentration is determined by the concentration of nucleoside triphosphates (dNTPs