Optimization of PCR amplification for sensitive capture of Methanopyrus isoleucyl-tRNA synthetase gene in environmental samples View Full Text


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Article Info

DATE

2010-07-27

AUTHORS

Zhiliang Yu

ABSTRACT

Microbes are abundant in marine and terrestrial ecosystems. However, the available evidence indicates that most microbial species in the environment cannot be cultivated. Accordingly, methodologies for characterizing genomes in environmental samples directly without cultivation have become foremost in importance. In this study, experimental conditions using PCR amplification to characterize such genomes were investigated with respect to the detection of target sequences present at low frequencies. Amplification of the isoleucyl-tRNA synthetase (IleRS) gene in Methanopyrus isolates was optimized regarding the choice of DNA polymerase, target sequence size, PCR primer size and the use of degenerate primers. Detection of IleRS was most sensitive when the target sequence size was about 1 kb. Among KOD, Taq and Pfu DNA polymerases, Pfu displayed the lowest efficiency, while KOD and Taq showed similar efficiency. Primers no shorter than 18mers gave satisfactory results. In addition, PCR bias caused by primer degeneracy could be alleviated by varying the balance between different added primers. Employing a combination of Taq DNA polymerase, a target DNA sequence approximately 1 kb in size, and primers of 20mer in length, PCR amplification of the IleRS gene could be achieved with as little as 2 pg pure isolated DNA template. More... »

PAGES

757-762

Identifiers

URI

http://scigraph.springernature.com/pub.10.1007/s13213-010-0097-1

DOI

http://dx.doi.org/10.1007/s13213-010-0097-1

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1040730348


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