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Ribotyping is a molecular technique for bacterial identification and characterization that uses information from rRNA-based phylogenetic analyses. [1] It is a rapid and specific method widely used in clinical diagnostics and analysis of microbial communities in food, water, and beverages.
In prokaryotes a small 30S ribosomal subunit contains the 16S ribosomal RNA. The large 50S ribosomal subunit contains two rRNA species (the 5S and 23S ribosomal RNAs). Therefore it can be deduced that in both bacteria and archaea there is one rRNA gene that codes for all three rRNA types :16S, 23S and 5S. [27] Bacterial 16S ribosomal RNA, 23S ...
Following the restriction reaction, the mixture of fragments is separated using either capillary or polyacrylamide electrophoresis in a DNA sequencer and the sizes of the different terminal fragments are determined by the fluorescence detector. Because the excised mixture of amplicons is analyzed in a sequencer, only the terminal fragments (i.e ...
Gel electrophoresis of large DNA or RNA is usually done by agarose gel electrophoresis. See the " chain termination method " page for an example of a polyacrylamide DNA sequencing gel. Characterization through ligand interaction of nucleic acids or fragments may be performed by mobility shift affinity electrophoresis .
A mobility shift assay is electrophoretic separation of a protein–DNA or protein–RNA mixture on a polyacrylamide or agarose gel for a short period (about 1.5-2 hr for a 15- to 20-cm gel). [4] The speed at which different molecules (and combinations thereof) move through the gel is determined by their size and charge, and to a lesser extent ...
Polyacrylamide gel electrophoresis with urea can also be used in RNA separation but it is most commonly used for fragmented RNA or microRNAs. [13] An RNA ladder is often run alongside the samples on an electrophoresis gel to observe the size of fragments obtained but in total RNA samples the ribosomal subunits can act as size markers. [11]
By using oligonucleotide primers targeted to conserved regions in the 16S and 23S genes, RISA fragments can be generated from most of the dominant bacteria in an environmental sample. While the majority of the rRNA operon serves a structural function, portions of the 16S-23S intergenic region can encode tRNAs depending on the bacterial species.
The plasmid is digested at a known restriction enzyme cut site downstream from the transcription start site such that the expected mRNA run-off product would be easily separated by gel electrophoresis. [1] [2] [4] DNA needs to be highly purified prior to running this assay.
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