Real-time PCR determination of rRNA gene copy number: absolute and relative quantification assays with Escherichia coli

Lee, Changsoo; Lee, Seungyong; Shin, Seung Gu; Hwang, Seokhwan

doi:10.1007/s00253-007-1300-6

Cited by 116 publications

(75 citation statements)

References 22 publications

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“…To determine whether reaction failures were due to inefficient heat lysis, inaccessibility of genomic DNA, or suboptimal PCR performance, we ran additional experiments in which a strain-specific fragment of the E. coli 16S gene was amplified in single E. coli cells using an optimized primer set (23). A total of 77 reactions were formulated using either single cells (N ¼ 62), approximately 100 cells (N ¼ 5), or cell suspension fluid containing no cells (N ¼ 10).…”

Section: Resultsmentioning

confidence: 99%

A programmable droplet-based microfluidic device applied to multiparameter analysis of single microbes and microbial communities

Leung

Zahn

Leaver

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

223

187

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We present a programmable droplet-based microfluidic device that combines the reconfigurable flow-routing capabilities of integrated microvalve technology with the sample compartmentalization and dispersion-free transport that is inherent to droplets. The device allows for the execution of user-defined multistep reaction protocols in 95 individually addressable nanoliter-volume storage chambers by consecutively merging programmable sequences of picoliter-volume droplets containing reagents or cells. This functionality is enabled by "flow-controlled wetting," a droplet docking and merging mechanism that exploits the physics of droplet flow through a channel to control the precise location of droplet wetting. The device also allows for automated cross-contaminationfree recovery of reaction products from individual chambers into standard microfuge tubes for downstream analysis. The combined features of programmability, addressability, and selective recovery provide a general hardware platform that can be reprogrammed for multiple applications. We demonstrate this versatility by implementing multiple single-cell experiment types with this device: bacterial cell sorting and cultivation, taxonomic gene identification, and high-throughput single-cell whole genome amplification and sequencing using common laboratory strains. Finally, we apply the device to genome analysis of single cells and microbial consortia from diverse environmental samples including a marine enrichment culture, deep-sea sediments, and the human oral cavity. The resulting datasets capture genotypic properties of individual cells and illuminate known and potentially unique partnerships between microbial community members.two-phase flow | droplet wetting | single-cell analysis | qPCR | environmental genomics M icrofluidic devices provide numerous advantages for biological analysis including automation, enhanced sensitivity and reaction efficiency in small volumes (1, 2), favorable mass transport properties (3, 4), and the potential for scalable and costeffective small volume assays (5). Indeed, advances in microfluidics over the past decade have resulted in increasingly sophisticated functionality and the emergence of two dominant and orthogonal strategies for fluid handling, based either on the use of integrated microvalves or the transport of microdroplets, both in closed channels or over electrode surfaces.The development of soft lithography (6) and the extension of this method to the fabrication of integrated microvalves using multilayer soft lithography (5) has enabled devices with thousands of active microvalves per cm 2 . This high level of integration enables device architectures capable of executing thousands of predefined "unit cell" reactions in parallel, with applications ranging from protein structure (4) and interaction studies (7,8) to single-cell analysis and genomics (2, 9, 10). Two-phase flow systems that manipulate picoliter (pL) volume droplets in closed channels have been shown to be ideally suited to high-speed serial analysis f...

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Section: Resultsmentioning

confidence: 99%

A programmable droplet-based microfluidic device applied to multiparameter analysis of single microbes and microbial communities

Leung

Zahn

Leaver

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

223

187

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“…This is a limitation of the GA approach that cannot be overcome without an absolute estimate. In contrast, absolute copy number estimates would need to be scaled to a known single-copy gene or to a concentration standard (such as in Lee et al 2008). …”

Section: Implications Of Rrna Copy Number Variation For Understandingmentioning

confidence: 99%

High‐throughput amplicon sequencing of rRNA genes requires a copy number correction to accurately reflect the effects of management practices on soil nematode community structure

2013

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Nematodes are abundant consumers in grassland soils, but more sensitive and specific methods of enumeration are needed to improve our understanding of how different nematode species affect, and are affected by, ecosystem processes. High‐throughput amplicon sequencing is used to enumerate microbial and invertebrate communities at a high level of taxonomic resolution, but the method requires validation against traditional specimen‐based morphological identifications. To investigate the consistency between these approaches, we enumerated nematodes from a 25‐year field experiment using both morphological and molecular identification techniques in order to determine the long‐term effects of annual burning and nitrogen enrichment on soil nematode communities. Family‐level frequencies based on amplicon sequencing were not initially consistent with specimen‐based counts, but correction for differences in rRNA gene copy number using a genetic algorithm improved quantitative accuracy. Multivariate analysis of corrected sequence‐based abundances of nematode families was consistent with, but not identical to, analysis of specimen‐based counts. In both cases, herbivores, fungivores and predator/omnivores generally were more abundant in burned than nonburned plots, while bacterivores generally were more abundant in nonburned or nitrogen‐enriched plots. Discriminate analysis of sequence‐based abundances identified putative indicator species representing each trophic group. We conclude that high‐throughput amplicon sequencing can be a valuable method for characterizing nematode communities at high taxonomic resolution as long as rRNA gene copy number variation is accounted for and accurate sequence databases are available.

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“…Plasmid abundance was determined by relative quantitative PCR. The plasmid replicon (Rep) was used as the target gene and a single-copy chromosomal dxs as the reference gene (19,20). BW25113⌬QAPRTase cells containing different plasmids were inoculated into cultures with relevant selection pressure, and total DNA was extracted from the cultures during the exponential phase, which was determined by periodic measurement of the OD 600 .…”

Section: Methodsmentioning

confidence: 99%

Novel Antibiotic-Free Plasmid Selection System Based on Complementation of Host Auxotrophy in the NAD De Novo Synthesis Pathway

Dong

Xiang

Shao

2010

Appl Environ Microbiol

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The use of antibiotic resistance genes in plasmids causes potential biosafety and clinical hazards, such as the possibility of horizontal spread of resistance genes or the rapid emergence of multidrug-resistant pathogens. This paper introduces a novel auxotrophy complementation system that allowed plasmids and host cells to be effectively selected and maintained without the use of antibiotics. An Escherichia coli strain carrying a defect in NAD de novo biosynthesis was constructed by knocking out the chromosomal quinolinic acid phosphoribosyltransferase (QAPRTase) gene. The resistance gene in the plasmids was replaced by the QAPRTase gene of E. coli or the mouse. As a result, only expression of the QAPRTase gene from plasmids can complement and rescue E. coli host cells in minimal medium. This is the first time that a vertebrate gene has been used to construct a nonantibiotic selection system, and it can be widely applied in DNA vaccine and gene therapy. As

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Real-time PCR determination of rRNA gene copy number: absolute and relative quantification assays with Escherichia coli

Cited by 116 publications

References 22 publications

A programmable droplet-based microfluidic device applied to multiparameter analysis of single microbes and microbial communities

A programmable droplet-based microfluidic device applied to multiparameter analysis of single microbes and microbial communities

High‐throughput amplicon sequencing of rRNA genes requires a copy number correction to accurately reflect the effects of management practices on soil nematode community structure

Novel Antibiotic-Free Plasmid Selection System Based on Complementation of Host Auxotrophy in the NAD De Novo Synthesis Pathway

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