The amplicon to be analysed must preferably correspond to a size of greater than 1000bp, purely for the sake of encountering a greater possibility of the restriction site. The amplicons must be preferably purified before digestion. This can be done by numerous commercially available purification kits. Care should be taken to choose the restriction enzymes. Certain restriction enzymes recognize the same sites, and cannot contribute productively to the analysis. Overnight digestion (10-16 hours) of about 300-500 ng of amplicon DNA in a 20 μL system with 4-5 units of Restriction Enzyme along with the recommended buffer at the prescribed temperature is recommended. Following digestion, the reaction is stopped and the entire digest run in a 2-3% agarose gel at 90-100V. The gel should be of a length that would allow proper resolution of minor fragments, as small as 100–200 bp.
Analysis of the patterns is done with methods used for RAPD patterns. Clusters of related bacteria can be represented in the form of a cladogram or phylogram. Initial analysis a multivariate analysis program such as NT-SYS or PAST furnishing details about the presence or absence of bands, marked as 1(for presence) and 0(for absence). The data can be subsequently used for generating a phylogram or cladogram. The data table can be used to plot a phylogenetic tree that would indicate the relationship of the organisms based on the restriction pattern obtained from their respective 16s genes.
Identification of Acinetobacter species isolated from clinical specimens by amplified ribosomal DNA restriction analysis
Jul 01, 2002; Background & objectives: Taxonomy of Acinetobacter has been changing ever since it was recognized to be associated with human...
Amplified ribosomal DNA restriction analysis of free-living bacteria present in the headbox of a Canadian paper machine.(Report)
Jul 01, 2009; Introduction The headbox of a paper machine is a tank that contains the papermaking materials. The mixture in the tank presents...