Quantitative multiplexing analysis of PCR-amplified ribosomal RNA genes by hierarchical oligonucleotide primer extension reaction.
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.
Wu
Jer-Horng
JH
Division of Environmental Science and Engineering, National University of Singapore, Singapore 117576.
Liu
Wen-Tso
WT
D001419:Bacteria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D001439:Bacteroides / Q000145:classification / Q000235:genetics; D017931:DNA Primers / Q000737:chemistry / Q000302:isolation & purification; D019075:Electrophoresis, Capillary; D004783:Environmental Microbiology; D005456:Fluorescent Dyes; D005798:Genes, Bacterial; D020459:Genes, rRNA; D010802:Phylogeny; D011061:Poly A / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics
D017931:DNA Primers; D005456:Fluorescent Dyes; D012336:RNA, Ribosomal, 16S; D011061:Poly A