INVESTIGATION OF TWO MOLECULAR BIOLOGY TECHNIQUES FOR THE STUDY OF MICROBIAL DIVERSITY

Abstract

Microbial diversity encompasses an immense number of prokaryotes which have been evolving on Earth over billions of years. Molecular phylogenetic analysis of evolutionarily conserved gene sequences, such as ribosomal RNA (rRNA), revolutionised bacterial classification and identification. Micro-organisms can now be identified by hybridisation using oligonucleotide probes targeting taxon-specific signatures carried by these genes. This approach is also commonly used in the resolution of microbial community structure. However, one study often needs multiple oligonucleotide probes with different specificities. Radio-labelling of each probe and separate or repeated hybridisations make this approach troublesome. Polymerase chain reaction (PCR) is another routinely used-technique in molecular identification of micro-organisms and study of microbial community. This technique is often used to selectively retrieve conserved gene fragments for the subsequent nucleotide sequence- or hybridisation-based analyses. However, PCR may generate two types of artefacts, namely chimeric molecule formation and preferential gene amplification, which may lead to erroneous assessment of microbial diversity. Chapter 3 describes the development of a highly efficient method for dot and slot blot hybridisations that simultaneously uses multiple oligonucleotide probes of different specificities in a single hybridisation chamber. This method saves significant time and reagents and minimises workers' exposure to radio-activities especially for studies that require many oligonucleotide probes. The method was tested successfully in identifying 27 actinomycete species from six different genera. It should provide a useful tool for rapid differentiation and identification of bacterial species as well as studies of microbial community structures. Chapter 4 and 5 describe a quantitative study of the frequencies of PCR-generated chimeric DNA molecules. A model was designed to quantify the frequency of chimera formation when pairs of homologous genes were co-amplified by PCR and determine the effects of several parameters such as sequence similarities of the input genes, the number of PCR cycles, the presence of damaged DNA and the length of the PCR elongation period. An approximate 30% occurrence of chimeric sequences after 30 cycles of co-amplification of two nearly identical 16S rRNA genes was detected and the frequency of chimera formation decreased to 12.9% and 14.7% for templates with 82% and 86% similarity, respectively. A positive correlation was found between the generation of chimeric molecules and the number of PCR cycles, and chimera formation exhibited increasingly high frequency in later cycles of amplification. Though damaged DNA, when present in a large amount, may contribute to chimera formation, strong evidences are obtained to demonstrate that prematurely terminated DNA strands during PCR are the main cause for chimera formation. A second model was used to assess the chimera formation as a result of PCR-amplification of 16S rRNA genes from mixed bacterial genomes. Comparative sequence analysis of 100 cloned PCR products detected a 32% occurrence of chimeric sequences after 30 cycles of amplification. Chimera formation was also shown to occur between different copies of rRNA genes within a micro-organism. Highly biased amplification of 16S rRNA genes was observed and such amplification was found to markedly increase the frequency of chimera formation occurring to the less amplified molecules. The results provide a quantitative assessment of the chimera formation as a consequence of PCR co-amplification of homologous genes. The high frequency of such artefacts demands cautious interpretation of results based on the analysis of conserved genes PCR-amplified from mixed genomes. With the ever wider application of PCR in direct isolation of 16S rRNA or other conserved genes from environmental DNA samples, the extent of chimeric molecule formation and its damaging consequences deserve some serious attention.