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Denaturing Polyacrylamide Gel Electrophoresis of DNA & RNA

The electrophoretic analysis of single stranded nucleic acids is complicated by the secondary structures assumed by these molecules. Separation on the basis of molecular weight requires the inclusion of denaturing agents which unfold the DNA or RNA strands and remove the influence of shape on their mobility. Nucleic acids form structures stabilized by hydrogen bonds between bases. Denaturing requires disrupting these hydrogen bonds. The most commonly used DNA denaturants are urea and formamide. Each of these forms hydrogen bonds with the DNA bases, "saturating" H-bond sites and preventing the formation of inter-base bonds.

Both formamide and urea effectively lower the melting point of the DNA molecules, allowing the structures to fall apart at lower temperatures. Generally, concentrations of urea or formamide are chosen to give melting temperatures around 50° C, and gels are run at that temperature. RNA is often denatured with harsher agents, because RNA tends to form stronger structures. RNA denaturation is discussed in RNA electrophoresis.

Denaturation of DNA by urea
 The denaturation of DNA by urea.

 

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