Please use this identifier to cite or link to this item:
Title: Quantitative multiplexing analysis of PCR-amplified ribosomal RNA genes by hierarchical oligonucleotide primer extension reaction
Authors: Wu, J.-H. 
Liu, W.-T. 
Issue Date: Jun-2007
Citation: Wu, J.-H., Liu, W.-T. (2007-06). Quantitative multiplexing analysis of PCR-amplified ribosomal RNA genes by hierarchical oligonucleotide primer extension reaction. Nucleic Acids Research 35 (11) : -. ScholarBank@NUS Repository.
Abstract: 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. © 2007 The Author(s).
Source Title: Nucleic Acids Research
ISSN: 03051048
DOI: 10.1093/nar/gkm413
Appears in Collections:Staff Publications

Show full item record
Files in This Item:
There are no files associated with this item.


checked on Nov 30, 2021


checked on Nov 23, 2021

Page view(s)

checked on Nov 18, 2021

Google ScholarTM



Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.