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When an electric field is applied, the DNA will begin to move through the gel, at a speed roughly inversely proportional to the length of the DNA molecule (shorter lengths of DNA travel faster) — this is the basis for size dependent separation in standard electrophoresis. In TGGE there is also a temperature gradient across the gel.
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]
In the earliest forms of denaturation mapping, DNA was denatured by heating in presence of formaldehyde [1] or glyoxal [3] and visualized using electron microscopy. Dyes that selectively bind to double stranded DNA like ethidium bromide could be used to monitor the extent of denaturation. But it was not possible to observe locations of ...
The process of DNA denaturation can be used to analyze some aspects of DNA. Because cytosine / guanine base-pairing is generally stronger than adenine / thymine base-pairing, the amount of cytosine and guanine in a genome is called its GC-content and can be estimated by measuring the temperature at which the genomic DNA melts. [ 2 ]
In the case of large DNA molecules, the DNA is frequently cut into smaller fragments using a DNA restriction endonuclease (or restriction enzyme). In other instances, such as PCR amplified samples, enzymes present in the sample that might affect the separation of the molecules are removed through various means before analysis.
Denaturation: If alkaline transfer methods are used, the DNA gel is placed into an alkaline solution (typically containing sodium hydroxide) to denature the double-stranded DNA. The denaturation in an alkaline environment may improve binding of the negatively charged thymine residues of DNA to a positively charged amino groups of membrane ...
Single-stranded DNA or RNA tends to fold up into molecules with complex shapes and migrate through the gel in a complicated manner based on their tertiary structure. Therefore, agents that disrupt the hydrogen bonds , such as sodium hydroxide or formamide , are used to denature the nucleic acids and cause them to behave as long rods again.
The DNA, however, is negatively charged at its phosphate groups and therefore can adsorb itself on the column. In order to make the adsorption possible, triethylammonium acetate (TEAA) is used. The positively charged ammonium ion of these molecules interacts with the DNA, and the alkyl chain with the hydrophobic surface of the solid phase.