Nucleic Acids Research Advance Access originally published online on June 6, 2007
Nucleic Acids Research 2007 35(11):e82; doi:10.1093/nar/gkm413
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Nucleic Acids Research, 2007, Vol. 35, No. 11 e82
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Quantitative multiplexing analysis of PCR-amplified ribosomal RNA genes by hierarchical oligonucleotide primer extension reaction
1Division of Environmental Science and Engineering, National University of Singapore, Singapore 117576 and 2Sustainable Environment Research Center, National Cheng Kung University, Taiwan, 701
*To whom correspondence should be addressed. Tel: +65-65161315; Fax: +65-67744202; Email: eseliuwt{at}nus.edu.sg
Received August 9, 2006. Revised May 4, 2007. Accepted May 4, 2007.
A method, termed hierarchical oligonucleotide primer extension (HOPE), is developed for quantitative, multiplexing detection of DNA targets present in PCR-amplified community 16S rRNA genes. It involves strand extension reaction and multiple oligonucleotide primers modified with different lengths of polyA at the 5' end and targeting 16S rRNA genes at different phylogenetic specificities. On annealing to the targets, these primers are extended with a single fluorescently labeled dideoxynucleoside triphosphate or a dye-terminator. Using a DNA autosequencer, these extended primers are separated and identified by size and dye color, and quantified and normalized based on the fluorescence intensities and internal size standards. Using a primer-to-target ratio >1000, constant primer extension efficiencies can be obtained with individual primers to establish a ‘calibration factor’ between individual primers and a universal or domain-specific primer, providing the relative abundance of targeted rRNA genes with respect to total rRNA genes. HOPE up to 10-plexing is demonstrated to correctly identify 20 different bacterial strains, and quantify different Bacteroides spp. in 16S rRNA gene amplicons from different model bacteria mixtures and the influent and effluent of a wastewater treatment plant. Single mismatch discrimination with detection sensitivity of a target down to 0.01–0.05% of total DNA template is achieved.
Present address: Jer-Horng Wu, Sustainable Environment Research Center, National Cheng Kung University, Taiwan, 701
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