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Abstract

The gene encoding the small subunit of the ribosomal RNA is commonly used for phylogenetic and diversity studies because of its universal distribution and its highly conserved sequence. While the 16S rRNA gene has usually a length of 1,500 bp it can reach up to 3,500 bp. This is due to the presence of self-splicing introns as was recently discovered in giant sulphur bacteria. These longer genes were not detected before because of a PCR-bias as well as following bioinformatics analysis. This study demonstrated the discrimination of longer DNA molecules in simultaneous amplification of two differently sized molecules mixed in different ratios. The kinetics of the reaction were implemented in a mathematical model and an identical amplification was simulated. Ratios between the two molecules were not maintained during the reaction in experimental and modelled amplification. The reason for the changes of template-to-amplicon ratios is the enzymatic limitation after a specific cycle. Bacterial diversity from samples containing a length heterogeneous mix of 16S rRNA genes, will be wrongly depicted due to the PCR-bias against longer genes. A method, that prevents the preferential amplification of shorter templates, could not have been established successfully in the course of this thesis. Nevertheless, the foundation for such a method have been laid and can be further investigated. Consequently, for molecular techniques and bioinformatics analysis, it is important to consider the possible occurrence of different 16S rRNA gene sizes and the PCR-bias towards shorter amplicons. High-throughput sequencing decreases the detection limited of elongated genes. The developed model can be used to calculate the template ratios of known 16S rRNA genes so that the natural abundance of the bacteria can be adjusted.