Browsing by Author "Baker, Gillian"
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Item 16 S rDNA primers and the unbiased assessment of thermophile diversity(Portland Press, 2004) Baker, Gillian; Cowan, Donald A.Our understanding of thermophile diversity is based predominantly on PCR studies of community DNA. ‘Universal’ and domain-specific rRNA gene PCR primers have historically been used for the assessment of microbial diversity without adequate regard to the degree of specificity of primer pairs to different prokaryotic groups. In a reassessment of the published primers commonly used for ‘universal’ and archaeal 16 S rDNA sequence amplification we note that substantial variations in specificity exist. An unconsidered choice of primers may therefore lead to significant bias in determination of microbial community composition. In particular, Archaea-specific primer sequences typically lack specificity for the Korarchaeota and Nanoarchaea and are often biased towards certain clades. New primer pairs specifically designed for ‘universal’ archaeal 16 S rDNA sequence amplification, with homology to all four archaeal groups, have been designed. Here we present the application of these new primers for preparation of 16 S libraries from thermophile communities.Item Metagenomics, gene discovery and the ideal biocatalyst(Portland Press, 2004) Cowan, Donald A.; Arslanoglu, A.; Burton, Stephanie G.; Cameron, Rory A.; Baker, Gillian; Smith, Jacques J.; Meyer, QuintonWith the rapid development of powerful protein evolution and enzyme-screening technologies, there is a growing belief that optimum conditions for biotransformation processes can be established without the constraints of the properties of the biocatalyst. These technologies can then be applied to find the ‘ideal biocatalyst’ for the process. In identifying the ideal biocatalyst, the processes of gene discovery and enzyme evolution play major roles. However, in order to expand the pool genes for in vitro evolution, new technologies, which circumvent the limitations of microbial culturability, must be applied. These technologies, which currently include metagenomic library screening, gene-specific amplification methods and even full metagenomic sequencing, provide access to a volume of ‘sequence space’ that is not addressed by traditional screening.Item PCR-based detection of non-indigenous microorganisms in ‘pristine’ environments(Elsevier, 2003) Baker, Gillian; Ah Tow, Lemese; Cowan, Donald A.PCR-based technologies are widely employed for the detection of specific microorganisms, and may be applied to the identification of non-indigenous microorganisms in ‘pristine’ environments. For ‘pristine’ environments such as those found on the Antarctic continent, the application of these methods to the assessment of environmental contamination from human activities must be treated with caution. Issues such as the possibility of non-human dispersal of organisms, stability and survival of non-indigenous organisms in vivo, the sensitivity, reproducibility and specificity of the PCR process (and particularly primer design) and the sampling regime employed must all be considered in detail. We conclude that despite these limitations, PCR and related technologies offer enormous scope for assessment of both natural and non-indigenous microbial distributions.Item Review and re-analysis of domain-specific 16S primers(Elsevier, 2003) Baker, Gillian; Smith, Jacques J.; Cowan, Donald A.The Polymerase Chain Reaction (PCR) has facilitated the detection of unculturable microorganisms in virtually any environmental source and has thus been used extensively in the assessment of environmental microbial diversity. This technique relies on the assumption that the gene sequences present in the environment are complementary to the “universal” primers used in their amplification. The recent discovery of new taxa with 16S rDNA sequences not complementary to standard universal primers suggests that current 16S rDNA libraries are not representative of true prokaryotic biodiversity. Here we re-assess the specificity of commonly used 16S rRNA gene primers and present these data in tabular form designed as a tool to aid simple analysis, selection and implementation. In addition, we present two new primer pairs specifically designed for effective ‘universal’ Archaeal 16S rDNA sequence amplification. These primers are found to amplify sequences from Crenarchaeote and Euryarchaeote type strains and environmental DNA.