| How does DGGE work? |
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During the first step, total DNA is extracted resulting in a complex mixture of DNA theoretically from all organisms present in the sample. Not only does this mixture contain DNA from all sample organisms, but each DNA molecule contains thousands of genes encoding various cellular functions. The amplification step, accomplished by polymerase chain reaction (PCR), selects a specific region of one gene and generates a multitude of copies of that gene segment. For most DGGE applications, the PCR step amplifies a highly variable region (V3) of the 16S rRNA gene found in all prokaryotes (bacteria and archaea). The DNA sequence of this region is what is used to classify an organism into a known family, genus, and species. Because PCR amplifies one specific gene segment, the amplification products are all the same length (size) but because these gene segments are amplified from all prokaryotes in the original sample, products will have different DNA sequences. Thus, the net result of the amplification step is the generation of a mixture of 16S rRNA gene segments representing all prokaryotes present in the original sample. During the next step, the amplification products (mixture of 16S rRNA gene segments) are separated on a denaturing gradient gel based on DNA sequence. To explore how separation is based on DNA sequences rather than size, examine each term in denaturing gradient gel electrophoresis Denaturing: breaking apart the two strands of the DNA molecule. Gradient Gel: gel with an increasing concentration of a chemical (denaturant) which breaks apart the DNA molecule. Electrophoresis: application of an electric current across a gel. In response to the current, double-stranded DNA migrates (moves down) the gel. Denaturing the DNA molecule forms Y- and T-shaped structures greatly slowing migration. DNA contains four nucleotide bases which bond across the two strands of the molecule – “G” forms three hydrogen bonds with “C”; “A” forms 2 hydrogen bonds with “T”. Thus, DNA segments with more GC base pairs (high GC content) form stronger bonds between the DNA strands than those with less GC base pairs. Consequently, high GC content DNA segments require a greater concentration of the denaturing chemical before the DNA strands break apart and migrate further down the gel.The mixture of 16S rRNA gene segments, each representing a type of organism in the original sample, is loaded at the top of the denaturing gradient gel. DNA migrates from the top (low denaturant concentration) toward the bottom of the gel (high denaturant concentration). DNA segments with low GC content denature near the top of gel and stop migrating. DNA segments with higher GC content denature further down the gel. DNA with the identical sequences migrate the same distance forming a “band”. Individual bands are excised for sequencing and results are compared to a database of 16S rRNA genes to identify the dominant organisms.
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