Denaturing Gradient Gel Electrophoresis involves PCR amplification of 16srRNA gene sequence. It involves the separation of amplicons of the same length but with different basepair sequences on polyacrylamide gel containing DNA denaturant.
Few steps in Denaturing Gradient Gel Electrophoresis steps are as follows
- DNA Extraction from Environmental samples.
- Amplification of DNA Fragments by PCR
- Incomplete denaturation and separation of amplicons containing DNA denaturant
- Staining and visualization of separated amplicon fragments.
Environmental samples are PCR amplified using16sRNA gene. Amplicons are amplified using primers containing GC clamp that prevent complete denaturation of DNA fragments. Amplification and analysis give rise to banding patterns. Each band corresponds to single species. After electrophoresis and staining bands can be excised from the gel followed by a clean-up procedure. They may be sequenced and further identification of the members of the community can be determined.
Thermal Gradient Gel Electrophoresis (TGGE) is a variation of Denaturing Gradient Gel Electrophoresis (DGGE). TGGE is the differentiating DNA based on the thermal properties of different nucleotide sequences that are of the same length. Amplification of the 16srDNA gene has been used in Molecular microbial ecology. Analysis of gels can be performed using software such as Phoenix ID, Applied Maths, Fingerprinting II Informatix. Similarity Indices can be based on the presence/absence of bands across the gel.
Analysis of soil microbial community in the environmental sample can be analyzed using UPMGA (unpaired mean group analysis). Analysis can be carried out using DGGE for the detection of sequence differences. Analysis can be done through the analysis of a small fragment of the 16sRNA gene.