Biomedical subjects

Jer-Horng Wu

Publications and source records attributed to Jer-Horng Wu.

4 recordsLinked to original sources

PubMed2006

Effects of target length on the hybridization efficiency and specificity of rRNA-based oligonucleotide microarrays.

The effect of target size on microarray hybridization efficiencies and specificity was investigated using a set of 166 oligonucleotide probes targeting the 16S rRNA gene of Escherichia coli. The targets included unfragmented native rRNA, fragmented rRNA ( approximately 20 to 100 bp), PCR amplicons (93 to 1,480 bp), and three synthetic single-stranded DNA oligonucleotides (45 to 56 bp). Fluorescence intensities of probes hybridized with targets were categorized into classes I (81 to 100% relative to the control probe), II (61 to 80%), III (41 to 60%), IV (21 to 40%), V (6 to 20%), and VI (0 to 5%). Good hybridization efficiency was defined for those probes conferring intensities in classes I to IV; those in classes V and VI were regarded as weak and false-negative signals, respectively. Using unfragmented native rRNA, 13.9% of the probes had fluorescence intensities in classes I to IV, whereas the majority (57.8%) exhibited false-negative signals. Similar trends were observed for the 1,480-bp PCR amplicon (6.6% of the probes were in classes I to IV). In contrast, after hybridization of fragmented rRNA, the percentage of probes in classes I to IV rose to 83.1%. Likewise, when DNA target sizes were reduced from 1,480 bp to 45 bp, this percentage increased approximately 14-fold. Overall, microarray hybridization efficiencies and specificity were improved with fragmented rRNA (20 to 100 bp), short PCR amplicons (<150 bp), and synthetic targets (45 to 56 bp). Such an understanding is important to the application of DNA microarray technology in microbial community studies.

DNA, Bacterial↗

PubMed2005

Emission characteristics of fluorescent labels with respect to temperature changes and subsequent effects on DNA microchip studies.

The effects of temperature, salt concentration, and formamide concentration on the emission characteristics of commonly used fluorescent labels were evaluated on DNA microchips. The emission intensities of different fluorophores without hybridization were observed to vary, each to a different extent, to mainly temperature changes. Rhodamine red, TAMRA (tetramethylrhodamine), and dyes from the carbocyanide group exhibited the largest variations, and Texas Red and Oregon Green exhibited the smallest variations. This temperature dependency was shown to affect results obtained during melting curve analysis in DNA microarray studies. To minimize the bias associated with the temperature-dependent emission of different fluorescent labels, a normalization step was proposed.

Fluorescein-5-isothiocyanate↗

PubMed2004

Evaluating single-base-pair discriminating capability of planar oligonucleotide microchips using a non-equilibrium dissociation approach.

The capability of planar rRNA-based oligonucleotide microarrays for single-base-pair discrimination was evaluated using an approach that compares the non-equilibrium dissociation profiles and dissociation temperatures (Tds) of all probe-target duplexes simultaneously. Three sets of 16S rRNA gene specific probes at different levels of specificity were used along with their counter probes for individual sets having either one or two mismatches (MM) to their targets at specific external (next to terminus) and various internal positions. Criteria based on the Td approach and a discrimination index (DI) were proven to be competent in discriminating PM from internal MM duplexes, but not always for external MM duplexes. Maximal DI for separating PM duplexes from ones with two and one internal MM usually occurred at temperatures approximately 5-10 degrees C and 10-15 degrees C, respectively, higher than the Tds of the PM duplexes. Washing buffer type and salt concentration, and MM number and position were shown statistically to affect dissociation profiles, Td, and single-base-pair discriminating capability. The reusability potential of the planar microchip was further demonstrated.

Base Pair Mismatch↗

PubMed2001

Characterization of microbial consortia in a terephthalate-degrading anaerobic granular sludge system.

The microbial composition and spatial distribution in a terephthalate-degrading anaerobic granular sludge system were characterized using molecular techniques. 16S rDNA clone library and sequence analysis revealed that 78.5% of 106 bacterial clones belonged to the delta subclass of the class Proteobacteria; the remaining clones were assigned to the green non-sulfur bacteria (7.5%), Synergistes (0.9%) and unidentified divisions (13.1%). Most of the bacterial clones in the delta-Proteobacteria formed a novel group containing no known bacterial isolates. For the domain Archaea, 81.7% and 18.3% of 72 archaeal clones were affiliated with Methanosaeta and Methanospirillum, respectively. Spatial localization of microbial populations inside granules was determined by transmission electron microscopy and fluorescent in situ hybridization with oligonucleotide probes targeting the novel delta-proteobacterial group, the acetoclastic Methanosaeta, and the hydrogenotrophic Methanospirillum and members of Methanobacteriaceae. The novel group included at least two different populations with identical rod-shape morphology, which made up more than 87% of the total bacterial cells, and were closely associated with methanogenic populations to form a nonlayered granular structure. This novel group was presumed to be the primary bacterial population involved in the terephthalate degradation in the methanogenic granular consortium.

Anaerobiosis↗