CROSS-REACTIVITY OF PROKARYOTIC 16S rRNA GENE-SPECIFIC PRIMERS WITH GENOMES FROM EUKARYOTIC ORGANISMS FROM MARSHLANDS

The 16S rRNA gene sequences have become the standard genetic markers for determining phylogenetic relationships among bacteria and archaea because of its presence in all prokaryotes. There has been conservation of structure and function of 16S rRNA gene and the length of the sequence (about 1500bp) in bacterial kingdom in spite of major evolutionary changes in other essential genes. Thus, it has become a gold standard in identification of non-culturable bacteria and archaea in environmental samples. Therefore, several 16S rRNA gene specific universal primers have been designed and are routinely used for identification of microbial diversity associated with environmental samples. But to target large microbial consortia universal primers need to be degenerate. It is generally believed that those primers do not give cross amplification with eukaryotic DNA. However, we have shown by in silico analysis, that they can cross react with many eukaryotic DNAs and give false results if that DNA is present in the environmental samples. We have validated our in silico findings using experimental approaches. Thus, cross-reactivity of these primers makes metagenomic studies more prone to false positive results and thereby decreases overall sensitivity.