Urea and formamide are used as denaturants in Denaturing Gradient Gel Electrophoresis (DGGE). In DGGE, urea, and formamide denature the DNA fragments by disrupting the Hydrogen bonding between the nitrogenous base pairs. As shown in the figure above, urea and formamide form a Hydrogen bond with nitrogenous bases which is why DNA fragments get denatured. Urea provides three Hydrogen bonding sites while formamide provides two H-bonding sites
Denaturing Gradient Gel Electrophoresis is an analytical tool that is most commonly used in the analysis of microbial communities. DGGE is used to separate DNA fragments based on their mobility under the increasing denaturing conditions created by different concentrations of urea and formamide. DGGE is applicable to separate DNA and RNA fragments but not the polypeptide chains. It is used to analyze the 16S ribosomal DNA of the bacterial samples during the study of the microbial population from a community.
DNA fragments having the same length of base pairs but different CG composition is called DNA sequence variants. DNA fragments may have the same lengths of base pairs but they may have a different composition of base pairs. The variance contributed by the differences in the composition of CG base pairs is the main basis that is considered during the DGGE analysis of these sequence variants.
DNA sequence variants have different melting points because they have different CG content and thus they migrate differently in Denaturing Gradient Gel Electrophoresis. It is assumed that each sequence variants of the same length of base pairs are from a different bacterial population within a community. So, if we see more than one band in DGGE then we should consider that there is more than one population within that community.
Theoretically, each band in the DGGE gel represents a single population of that community. However, it is nested PCR products that are used for separating through the DGGE, not the long PCR product. This is because nested PCR reduces the non-specific binding of the PRC products contributed by the unexpected primer binding sites. Therefore, nested PRC products are more specific than long PCR products and are the most suitable for DGGE analysis.
This way, urea and formamide act as denaturants and disrupt the Hydrogen bonding among the nitrogenous base pairs and denature the double-stranded DNA fragments aiding their movement in Density Gradient Gel Electrophoresis.
