What is denaturation and re-association DNA

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Denaturation - renaturation

The DNA strands can be separated from one another by heating or adding alkali. The thermal denaturation dissolves all interactions between the bases at the same time. A cooperative melting occurs, which can be followed particularly well by spectroscopy. Similar to the determination of the melting point of crystalline substances, when the DNA melts at a certain temperature, the double-helical structure suddenly collapses.

The double helix melts into two single strands. The temperature at which half of the DNA is single-stranded is called T.m designated. The melting process can be demonstrated by UV spectroscopy, because the formation of the stacking interactions is associated with a gain in energy and the total absorption of the polymer is 1.4 times weaker than the absorption of the same molar amount of individual nucleotides. With the help of UV spectroscopy it is easy to create stability / melting profiles for genomic DNA. The stability of DNA is proportional to its CG content. In microbiology in particular, attempts have been made to classify different organisms. Even thermostable organisms, however, always contain a minimum amount of adenosine and thymidine. The melting analyzes alone cannot explain why there are living things that have an optimum growth rate of> 106 ° C. Many other factors play an additional role here.

Not only the melting profile of a DNA strand is characteristic, but also the profile of the reassociation. This is often referred to as reannealing. If the denatured DNA is cooled again, the complementary strands find each other again. The speed of reassociation depends on various factors. The concentration of the cations to buffer the negative charge on the backbone is the temperature, at best 25 ° C below Tm and the size and concentration of the DNA strands are of particular importance. In addition, so-called repetitive sequences accelerate reassociation. Reassociation is a two-step process. The first step is nucleation. It describes how the first complementary base pairs come together. This step is the slower and therefore the speed-determining step. The second, faster step is to create the remaining base pairs.

While naturally occurring DNA always has a very high melting point of at least 85 ° C, synthetic DNAs show melting points that are approximately proportional to the GC content and chain length up to a chain length of 50 base pairs (bp). So-called breathing occurs at the open ends, because the ends open and close permanently. The melting behavior of so-called oligonucleotides plays a decisive role in the polymerase chain reaction (PCR) and is discussed in detail there.

Denaturing-renaturing can be used for pedigree certificates. Here, DNA from two different species is melted and the single strands are combined in a reaction vessel for reassociation. It is then evaluated how many hybrids have formed. The frequency of hybrids is a measure of the relationship between the two organisms. This approach can of course also be carried out for DNA segments only. However, this method is rarely used today because direct DNA sequencing allows much more precise analyzes.

The addition of alkali is a particularly effective method of quickly and completely separating the DNA strands. The protons that form the hydrogen bonds between the bases are withdrawn from the DNA. The bases lose their quasi-aromatic structure and reduce the electrons in the stacking interactions. The stacking forces are no longer sufficient to compensate for the repulsion of the negatively charged sugar-phosphate backbone. An extremely rapid strand separation occurs, which is used in the so-called alkaline lysis for plasmid isolation.