How is 16s rRNA extracted and used to determine microbial diversity?


The molecular analysis of ribosomal RNA (rRNA), specifically the 16s sub unit is crucial to examining the diversity of microorganisms. The sequences of the 16s rRNA provides insight into the lineage of prokaryotic organisms and can provide information of the relative abundance of specific microbial communities. But how is it we can look at the specific sequence of rRNA from a microbial source such as meat or water.
The methods for DNA and RNA extraction involve a combination of lysis and extractions to yield a crude DNA product. We can then amplify the rRNA gene with the polymerase chain reaction (PCR) using primers specifc for the 16s rRNA gene fragment. Additionally the RNA from cells can be separated and examined for the rRNA product. Separation of DNA from RNA is possible by gel filtration chromatography because of the structural differences of DNA and RNA ( double stranded vs single stranded, T vs U)
Using a technique known as reverse transcription polymerase chain reaction (RT-PCR) which uses a reverse transcriptase enzyme we can reverse transcribe the rRNA molecule into its complementary DNA (cDNA) The genetic fingerprint of both PCR and RT-PCR products can be determined by a denaturing gradient gel electrophoresis (DGGE).

DGGE allows researchers and clinical scientists to amplify the 16s RNA from a source: meat or water, and visualize the variations in the microbial communities. The banding pattern in the gel provides high enough resolution to determine specific changes in sequence which can give information of mutations and help determine genetic diversity. Additionally the intensity of the banding patterns will provide and idea of the abundance of certain microbial communities.



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