Development of a novel method for operating magnetic particles, Magtration Technology, and its use for automating nucleic acid purification

Obata, Kimimichi; Segawa, Osamu; Yakabe, Mitsuru; Ishida, Yasumasa; Kuroita, Toshihiro; Ikeda, Katsunori; Kawakami, Bunsei; Kawamura, Y.; Yohda, Masafumi; Matsunaga, Tadashi; Tajima, Hideji

doi:10.1016/s1389-1723(01)80280-2

Cited by 57 publications

(14 citation statements)

References 6 publications

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“…Extraction of DNA Total DNA was extracted using a fully automated nucleic acid extractor (Magtration System 6GC, Precision System Science, Chiba, Japan) using magnetic bead technology [16,24]. An aliquot (1 mL) of the sample was centrifuged at 14,0009g for 10 min and the supernatant was decanted.…”

Section: Bioreactor Operationmentioning

confidence: 99%

Use of order-specific primers to investigate the methanogenic diversity in acetate enrichment system

Shin

Lee

Hwang

et al. 2008

J Ind Microbiol Biotechnol

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The applicability of order-specific primers in minimizing the possible underestimation of microbial diversity was evaluated via denaturing gradient gel electrophoresis (DGGE) analysis of a lab-scale anaerobic digester. Initially, a population analysis with real-time quantitative PCR demonstrated the existence of three methanogenic orders--Methanobacteriales, Methanomicrobiales, and Methanosarcinales--throughout the reaction period. DGGE analyses with three pairs of order-specific primers yielded eight operational taxonomic units (OTUs), whereas DGGE analysis with two independent Archaea-specific primers identified only five. Moreover, the order-specific primers amplified at least one OTU affiliated with each order, whereas no members of Methanobacteriales or Methanomicrobiales were identified with Archaea-specific primers in most samples. These findings provide evidence that order-specific analysis can detect the diversity of methanogens in greater detail than conventional Archaea-specific analysis.

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Section: Bioreactor Operationmentioning

confidence: 99%

Use of order-specific primers to investigate the methanogenic diversity in acetate enrichment system

Shin

Lee

Hwang

et al. 2008

J Ind Microbiol Biotechnol

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“…Afullyautomatednucleicacidextractor(MagtrationSystem 6GC, PSS Co., Chiba, Japan) employing magnetic bead technology (Obata et al, 2001) was used to extract and purify genomic DNA from thevarioussources (i.e.,nine purecultures, batch experiment, and three anaerobic processes). The automated instrumentation has been reported to extract DNA with a consistent efficiency and high purity by eliminating the stepsofmanualpreparationthatmaycausecrosscontamination (Grisold et al, 2002;Yu et al, 2005).…”

Section: Extraction Of Genomic Dnamentioning

confidence: 99%

Use of real‐time PCR for group‐specific quantification of aceticlastic methanogens in anaerobic processes: Population dynamics and community structures

Kim²,

Hwang

2006

Biotech & Bioengineering

139

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The TaqMan quantitative PCR (QPCR) method was used to detect and quantify the 16S rRNA genes of aceticlastic methanogens at different taxonomic levels. Three different sets of primers coupled with a TaqMan probe for QPCR assays to detect the 16S rRNA genes of the order Methanosarcinales, as well as the families Methanosarcinaceae and Methanosaetaceae, were separately used. Using these primer and probe sets, the 16S rRNA genes of aceticlastic methanogens in samples from various anaerobic processes (i.e., nine pure cultures, batch experiment, and three different continuous processes including a full-scale digester), were monitored and quantified by QPCR assays. A batch experiment cultivating a mixture of aceticlastic methanogens, was conducted to monitor their population dynamics. Using this group-specific quantification method, the dynamics of a competition between two aceticlastic populations, as modulated by the acetate concentration, could well be described. The target 16S rRNA genes in environmental samples, collected from three different anaerobic processes treating sludge, cheese whey, and synthetic wastewaters, were additionally quantified. The quantified 16S rRNA gene concentrations for all samples successfully represented the community structures of the target methanogens, which were correlated accurately with the operational parameters of the anaerobic processes. It was also successful to demonstrate probe nesting of aceticlastic methanogens at the levels of order and family.

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“…As described previously (Yu et al, 2005), a fully automated nucleic acid extractor (Magtration System 6GC, PSS Co., Chiba, Japan) employing magnetic bead separation technique (Obata et al, 2001) was used to extract and purify genomic DNA from the various sources (i.e., 16 purchased strains, 7 environmental clones). Purified DNA was stored at À208C until used for subsequent experiments.…”

Section: Sources and Extraction Of Dnamentioning

confidence: 99%

Primer and probe sets for group‐specific quantification of the genera Nitrosomonas and Nitrosospira using real‐time PCR

Lim

Shin

et al. 2007

Biotech & Bioengineering

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Use of quantitative real-time PCR (QPCR) with TaqMan probes is increasingly popular in various environmental works to detect and quantify a specific microorganism or a group of target microorganism. Although many aspects of conducting a QPCR assay have become very easy to perform, a proper design of oligonucleotide sequences comprising primers and a probe is still considered as one of the most important aspects of a QPCR application. This work was conducted to design group specific primer and probe sets for the detection of ammonia oxidizing bacteria (AOB) using a real-time PCR with a TaqMan system. The genera Nitrosomonas and Nitrosospira were grouped into five clusters based on similarity of their 16S rRNA gene sequences. Five group-specific AOB primer and probe sets were designed. These sets separately detect four subgroups of Nitrosomonas (Nitrosomonas europaea-, Nitrosococcus mobilis-, Nitrosomonas nitrosa-, and Nitrosomonas cryotolerans-clusters) along with the genus Nitrosospira. Target-group specificity of each primer and probe set was initially investigated by analyzing potential false results in silico, followed by a series of experimental tests for QPCR efficiency and detection limit. In general, each primer and probe set was very specific to the target group and sensitive to detect target DNA as low as two 16S rRNA gene copies per reaction mixture. QPCR efficiency, higher than 93.5%, could be achieved for all primer and probe sets. The primer and probe sets designed in this study can be used to detect and quantify the beta-proteobacterial AOB in biological nitrification processes and various environments.

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Development of a novel method for operating magnetic particles, Magtration Technology, and its use for automating nucleic acid purification

Cited by 57 publications

References 6 publications

Use of order-specific primers to investigate the methanogenic diversity in acetate enrichment system

Use of order-specific primers to investigate the methanogenic diversity in acetate enrichment system

Use of real‐time PCR for group‐specific quantification of aceticlastic methanogens in anaerobic processes: Population dynamics and community structures

Primer and probe sets for group‐specific quantification of the genera Nitrosomonas and Nitrosospira using real‐time PCR

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