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Capillary electrophoresis of ribosomal RNA for characterisation of microbial communities


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Nai, YH 2013 , 'Capillary electrophoresis of ribosomal RNA for characterisation of microbial communities', PhD thesis, University of Tasmania.

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This thesis documents research on new capillary electrophoresis (CE) based rRNA
fingerprint approaches for characterisation of low diversity microbial communities.
In the first body of work, an alternative approach for sieving polymer synthesis through
reversible addition fragmentation chain transfer (RAFT) polymerisation is presented.
Sieving polymer matrices are typically synthesised by conventional free radical
polymerisation. This thesis describes the first synthesis of a high molecular weight
poly(n,n-dimethylacrylamide) (PDMA) in which both the molar mass and
polydispersity distribution were controlled by RAFT polymerisation. A multi-step chain
extension is detailed and the physical properties and separation performance of
DNA/RNA using this RAFT polymer are described.
The second body of work deals with the development of new approach for
characterisation of microbial communities using CE. The new approach involves
conformational separation of microbial 16S ribosomal RNA (rRNA) molecules
containing the highly variable regions present in 16S rRNA. Single stranded
conformation polymorphism (SSCP) is a separation technique based on the principle
that for nucleic acid fragments of equal lengths, variation in sequences can affect
nucleic acid folding and hence can be separated due to the difference in electrophoretic
mobility. While CE DNA-SSCP has been commonly applied in clinical mutation
diagnostic tests and studies of microbial diversity, CE rRNA-SSCP has yet to be
demonstrated. In this work, an enzymatic based RNA-oligonucleotide cleavage method
was employed to cleave the 16S rRNA (~1542 bases) to smaller fragments of similar
length (~340 bases). This strategy uses a eubacterial ‘scissor’ probe to target and hybridise highly conserved sites within the rRNA flanking highly variable regions (e.g.
V1, V2 or V3). As rRNA is synthesised only by actively-growing cells, together with its
role as the marker molecule for assigning sequences to genera and species, it can thus
be used to correlate to the functioning members of microbial communities. Taking
advantage of these unique properties, CE-rRNA-SSCP circumvents the need for
polymerase chain reaction (PCR) amplification and retains the quantitative information
regarding to the evenness of the microbial community that is important for ecological
studies that were otherwise lost during PCR step. Compared to gel electrophoresis based
approach, CE- rRNA SSCP significantly decreased the analysis time from 24 hours to
60 min and the use of a fluorescently labelled hybridisation probe for detection
decreased the sample requirement by ten-fold. The combination of fast analysis time,
low sample requirement and sensitive fluorescence detection makes CE-rRNA-SSCP an
appealing new approach for characterising low diversity microbial communities.
The third body of work deals with the conception and development of a novel
characterisation approach termed multiplex cleavage microbial community analysis
(MCMCA), which is a potential method to simultaneously link the phylogeny of
multiple groups of metabolically active microorganisms to their respective metabolic
activity and relative abundance within a community. MCMCA utilizes the similar
sequence-specific cleavage of rRNA molecules with oligonucleotides and RNase H
employed in previous approach but differs by the use of multiple taxon specific probes
selected to specifically cut the 16S rRNA into discrete fragments varying in length. The
cleaved rRNA mixture is subsequently mixed with a fluorescently labelled locked
nucleic acid (LNA) universal hybridisation probe and resolved using denaturing CE size
separation. The feasibility of this rational is tested using model microbial strains, followed by optimisation of the cleavage procedure to achieve multiplex cleavage in a
model microbial community. This approach was then applied to characterise a
hydrocarbon degrading enrichment community derived from soil.

Item Type: Thesis - PhD
Authors/Creators:Nai, YH
Keywords: Separation Science, Capillary electrophoresis, Ribosomal RNA, Microbiology, Community fingerprinting
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