Acquisition of 16S rrNA gene sequence

The DNA fragments containing all or part of the 16s rRNA gene are commonly obtained through PCR. The primers are designed to anneal to the conserved regions within the gene and sometimes this enables the use of one primer group to amplify 16S from several phylogenetically diverse bacteria.  However, no one set of primers can amplify all the genes from all the Archaea and all the Bacteria and many primer sets have been designed which are phylum- or group-specific. The use of combinations of these collection means that most known microorganisms can be amplified from any pure culture, environmental or mixed culture.

While acquisition of sequence through PCR is quick, there are limitations imposed through the method itself. PCR can generate chimeras, PCR products which are composed of the 5’ end of one species’ gene coupled to the 3’ end of another. while computer programs exist  to eliminate these false  sequences from  the  last  results it is sometimes hard to detect them when dealing with  undiscovered or rare organisms. The existence of the division Korarchaeota in the kingdom Archaea was in doubt for precisely this reason. Ten years of research into how to cultivate the organism confirmed which it did indeed form a deeply branched division of the Archaea.