scholarly journals 16S rRNA probe design for HCR-FISH v1 (protocols.io.wcefate)

protocols.io ◽  
2018 ◽  
Author(s):  
Kayley Hake ◽  
Nicole King
Keyword(s):  
16S Rrna ◽  
Probe Design

scholarly journals A MCMC strategy for group-specific 16S rRNA probe design

2009 ◽  
Vol 02 (06) ◽  
pp. 412-418
Author(s):  
Yi-Bo Wu ◽  
Li-Rong Yan ◽  
Hui Liu ◽  
Han-Chang Sun ◽  
Hong-Wei Xie
Keyword(s):  
16S Rrna ◽  
Probe Design

Differential sensitivity of 16S rRNA targeted oligonucleotide probes used for fluorescence in situ hybridization is a result of ribosomal higher order structure

1996 ◽  
Vol 42 (10) ◽  
pp. 1061-1071 ◽  
Author(s):  
Marc E. Frischer ◽  
Peter J. Floriani ◽  
Sandra A. Nierzwicki-Bauer
Keyword(s):  
In Situ Hybridization ◽  
16S Rrna ◽  
Fluorescence In Situ Hybridization ◽  
Differential Sensitivity ◽  
Probe Design ◽  
Oligonucleotide Probes ◽  
Whole Cells ◽  
Gene Probes ◽  
Wide Range

The use of 16S rRNA targeted gene probes for the direct analysis of microbial communities has revolutionized the field of microbial ecology, yet a comprehensive approach for the design of such probes does not exist. The development of 16S rRNA targeted oligonucleotide probes for use with fluorescence in situ hybridization (FISH) procedures has been especially difficult as a result of the complex nature of the rRNA target molecule. In this study a systematic comparison of 16S rRNA targeted oligonucleotide gene probes was conducted to determine if target location influences the hybridization efficiency of oligonucleotide probes when used with in situ hybridization protocols for the detection of whole microbial cells. Five unique universal 12-mer oligonucleotide sequences, located at different regions of the 16S rRNA molecule, were identified by a computer-aided sequence analysis of over 1000 partial and complete 16S rRNA sequences. The complements of these oligomeric sequences were chemically synthesized for use as probes and end labeled with either [γ-32P] ATP or the fluorescent molecule tetramethylrhodamine-5/-6. Hybridization sensitivity for each of the probes was determined by hybridization to heat-denatured RNA immobilized on blots or to formaldehyde fixed whole cells. All of the probes hybridized with equal efficiency to denatured RNA. However, the probes exhibited a wide range of sensitivity (from none to very strong) when hybridized with whole cells using a previously developed FISH procedure. Differential hybridization efficiencies against whole cells could not be attributed to cell wall type, since the relative probe efficiency was preserved when either Gram-negative or -positive cells were used. These studies represent one of the first attempts to systematically define criteria for 16S rRNA targeted probe design for use against whole cells and establish target site location as a critical parameter in probe design.Key words: 16S rRNA, oligonucleotide probes, in situ hybridization.

scholarly journals Genome-Based Targeted Sequencing as a Reproducible Microbial Community Profiling Assay

mSphere ◽  
2021 ◽  
Vol 6 (2) ◽  
Author(s):  
Jacquelynn Benjamino ◽  
Benjamin Leopold ◽  
Daniel Phillips ◽  
Mark D. Adams
Keyword(s):  
16S Rrna ◽  
Microbial Communities ◽  
Relative Abundance ◽  
Primer Extension ◽  
New Method ◽  
Probe Design ◽  
Primer Extension Reaction ◽  
Community Profiling ◽  
Single Primer ◽  
Reference Genomes

ABSTRACT Current sequencing-based methods for profiling microbial communities rely on marker gene (e.g., 16S rRNA) or metagenome shotgun sequencing (mWGS) analysis. We present an approach based on a single-primer extension reaction using a highly multiplexed oligonucleotide probe pool. This approach, termed MA-GenTA (microbial abundances from genome tagged analysis), enables quantitative, straightforward, cost-effective microbiome profiling that combines desirable features of both 16S rRNA and mWGS strategies. The use of multiple probes per target genome and rigorous probe design criteria enabled robust determination of relative abundance. To test the utility of the MA-GenTA assay, probes were designed for 830 genome sequences representing bacteria present in mouse stool specimens. Comparison of the MA-GenTA data with mWGS data demonstrated excellent correlation down to 0.01% relative abundance and a similar number of organisms detected per sample. Despite the incompleteness of the reference database, nonmetric multidimensional scaling (NMDS) clustering based on the Bray-Curtis dissimilarity metric of sample groups was consistent between MA-GenTA, mWGS, and 16S rRNA data sets. MA-GenTA represents a potentially useful new method for microbiome community profiling based on reference genomes. IMPORTANCE New methods for profiling the microbial communities can create new approaches to understanding the composition and function of those communities. In this study, we combined bacterial genome-specific probe design with a highly multiplexed single primer extension reaction as a new method to profile microbial communities, using stool from various mouse strains as a test case. This method, termed MA-GenTA, was benchmarked against 16S rRNA gene sequencing and metagenome sequencing methods and delivered similar relative abundance and clustering data. Since the probes were generated from reference genomes, MA-GenTA was also able to provide functional pathway data for the stool microbiome in the assayed samples. The method is more informative than 16S rRNA analysis while being less costly than metagenome shotgun sequencing.

scholarly journals Microarray Analysis and Barcoded Pyrosequencing Provide Consistent Microbial Profiles Depending on the Source of Human Intestinal Samples

2011 ◽  
Vol 77 (6) ◽  
pp. 2071-2080 ◽  
Author(s):  
Bartholomeus van den Bogert ◽  
Willem M. de Vos ◽  
Erwin G. Zoetendal ◽  
Michiel Kleerebezem
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Microarray Analysis ◽  
High Throughput ◽  
Large Scale ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Probe Design ◽  
Rrna Gene ◽  
Small Intestinal

ABSTRACTLarge-scale and in-depth characterization of the intestinal microbiota necessitates application of high-throughput 16S rRNA gene-based technologies, such as barcoded pyrosequencing and phylogenetic microarray analysis. In this study, the two techniques were compared and contrasted for analysis of the bacterial composition in three fecal and three small intestinal samples from human individuals. As PCR remains a crucial step in sample preparation for both techniques, different forward primers were used for amplification to assess their impact on microbial profiling results. An average of 7,944 pyrosequences, spanning the V1 and V2 region of 16S rRNA genes, was obtained per sample. Although primer choice in barcoded pyrosequencing did not affect species richness and diversity estimates, detection ofActinobacteriastrongly depended on the selected primer. Microbial profiles obtained by pyrosequencing and phylogenetic microarray analysis (HITChip) correlated strongly for fecal and ileal lumen samples but were less concordant for ileostomy effluent. Quantitative PCR was employed to investigate the deviations in profiling between pyrosequencing and HITChip analysis. Since cloning and sequencing of random 16S rRNA genes from ileostomy effluent confirmed the presence of novel intestinal phylotypes detected by pyrosequencing, especially those belonging to theVeillonellagroup, the divergence between pyrosequencing and the HITChip is likely due to the relatively low number of available 16S rRNA gene sequences of small intestinal origin in the DNA databases that were used for HITChip probe design. Overall, this study demonstrated that equivalent biological conclusions are obtained by high-throughput profiling of microbial communities, independent of technology or primer choice.

A parallel computing algorithm for 16S rRNA probe design

2006 ◽  
Vol 66 (12) ◽  
pp. 1546-1551 ◽  
Author(s):  
Dianhui Zhu ◽  
Yuriy Fofanov ◽  
Richard C. Willson ◽  
George E. Fox
Keyword(s):  
Parallel Computing ◽  
16S Rrna ◽  
Probe Design ◽  
Computing Algorithm

scholarly journals 16S rRNA Gene-Based Oligonucleotide Microarray for Environmental Monitoring of the Betaproteobacterial Order “Rhodocyclales”

2005 ◽  
Vol 71 (3) ◽  
pp. 1373-1386 ◽  
Author(s):  
Alexander Loy ◽  
Claudia Schulz ◽  
Sebastian Lücker ◽  
Andreas Schöpfer-Wendels ◽  
Kilian Stoecker ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Activated Sludge ◽  
Treatment Plant ◽  
Pcr Amplification ◽  
Oligonucleotide Microarray ◽  
Probe Design ◽  
Rrna Gene ◽  
Microarray Hybridization

ABSTRACT For simultaneous identification of members of the betaproteobacterial order “Rhodocyclales” in environmental samples, a 16S rRNA gene-targeted oligonucleotide microarray (RHC-PhyloChip) consisting of 79 probes was developed. Probe design was based on phylogenetic analysis of available 16S rRNA sequences from all cultured and as yet uncultured members of the “Rhodocyclales.” The multiple nested probe set was evaluated for microarray hybridization with 16S rRNA gene PCR amplicons from 29 reference organisms. Subsequently, the RHC-PhyloChip was successfully used for cultivation-independent “Rhodocyclales” diversity analysis in activated sludge from an industrial wastewater treatment plant. The implementation of a newly designed “Rhodocyclales”-selective PCR amplification system prior to microarray hybridization greatly enhanced the sensitivity of the RHC-PhyloChip and thus enabled the detection of “Rhodocyclales” populations with relative abundances of less than 1% of all bacteria (as determined by fluorescence in situ hybridization) in the activated sludge. The presence of as yet uncultured Zoogloea-, Ferribacterium/Dechloromonas-, and Sterolibacterium-related bacteria in the industrial activated sludge, as indicated by the RHC-PhyloChip analysis, was confirmed by retrieval of their 16S rRNA gene sequences and subsequent phylogenetic analysis, demonstrating the suitability of the RHC-PhyloChip as a novel monitoring tool for environmental microbiology.

Protein-induced conformational changes in 16S rRNA during assembly of the Escherichia Coli 30S ribosomal subunit: A STEM study

1987 ◽  
Vol 45 ◽  
pp. 910-911
Author(s):  
M. Boublik ◽  
V. Mandiyan ◽  
J.F. Hainfeld ◽  
J.S. Wall
Keyword(s):  
16S Rrna ◽  
Conformational Changes ◽  
Ribosomal Proteins ◽  
Structural Changes ◽  
Ribosomal Subunit ◽  
Compact Structure ◽  
E Coli ◽  
Freeze Dried ◽  
16S Rna ◽  
30S Subunit

The aim of this study is to understand the mechanism of 16S rRNA folding into the compact structure of the small 30S subunit of E. coli ribosome. The assembly of the 30S E. coli ribosomal subunit is a sequence of specific interactions of 16S rRNA with 21 ribosomal proteins (S1-S21). Using dedicated high resolution STEM we have monitored structural changes induced in 16S rRNA by the proteins S4, S8, S15 and S20 which are involved in the initial steps of 30S subunit assembly. S4 is the first protein to bind directly and stoichiometrically to 16S rRNA. Direct binding also occurs individually between 16S RNA and S8 and S15. However, binding of S20 requires the presence of S4 and S8. The RNA-protein complexes are prepared by the standard reconstitution procedure, dialyzed against 60 mM KCl, 2 mM Mg(OAc)2, 10 mM-Hepes-KOH pH 7.5 (Buffer A), freeze-dried and observed unstained in dark field at -160°.

Imaging of ribosomal RNA-protein interactions by dark-field STEM

1989 ◽  
Vol 47 ◽  
pp. 250-251
Author(s):  
M. Boublik ◽  
V. Mandiyan ◽  
S. Tumminia ◽  
J.F. Hainfeld ◽  
J.S. Wall
Keyword(s):  
Nucleic Acid ◽  
16S Rrna ◽  
Dark Field ◽  
Radius Of Gyration ◽  
Central Domain ◽  
The Core ◽  
16S Rna ◽  
30S Subunit ◽  
Core Particles

Success in protein-free deposition of native nucleic acid molecules from solutions of selected ionic conditions prompted attempts for high resolution imaging of nucleic acid interactions with proteins, not attainable by conventional EM. Since the nucleic acid molecules can be visualized in the dark-field STEM mode without contrasting by heavy atoms, the established linearity between scattering cross-section and molecular weight can be applied to the determination of their molecular mass (M) linear density (M/L), mass distribution and radius of gyration (RG). Determination of these parameters promotes electron microscopic imaging of biological macromolecules by STEM to a quantitative analytical level. This technique is applied to study the mechanism of 16S rRNA folding during the assembly process of the 30S ribosomal subunit of E. coli. The sequential addition of protein S4 which binds to the 5'end of the 16S rRNA and S8 and S15 which bind to the central domain of the molecule leads to a corresponding increase of mass and increased coiling of the 16S rRNA in the core particles. This increased compactness is evident from the decrease in RG values from 114Å to 91Å (in “ribosomal” buffer consisting of 10 mM Hepes pH 7.6, 60 mM KCl, 2 m Mg(OAc)2, 1 mM DTT). The binding of S20, S17 and S7 which interact with the 5'domain, the central domain and the 3'domain, respectively, continues the trend of mass increase. However, the RG values of the core particles exhibit a reverse trend, an increase to 108Å. In addition, the binding of S7 leads to the formation of a globular mass cluster with a diameter of about 115Å and a mass of ∽300 kDa. The rest of the mass, about 330 kDa, remains loosely coiled giving the particle a “medusa-like” appearance. These results provide direct evidence that 16S RNA undergoes significant structural reorganization during the 30S subunit assembly and show that its interactions with the six primary binding proteins are not sufficient for 16S rRNA coiling into particles resembling the native 30S subunit, contrary to what has been reported in the literature.

Effects of a Supplemental Spanish Phonological Awareness Intervention on Latinx Preschoolers' Dual Language Emergent Literacy Skills

2020 ◽  
Vol 29 (3) ◽  
pp. 1283-1300
Author(s):  
Xigrid T. Soto ◽  
Andres Crucet-Choi ◽  
Howard Goldstein
Keyword(s):  
At Risk ◽  
Phonological Awareness ◽  
Emergent Literacy ◽  
Language Learners ◽  
Literacy Instruction ◽  
Dual Language ◽  
Literacy Skills ◽  
Dual Language Learners ◽  
Probe Design ◽  
Emergent Literacy Skills

Purpose Preschoolers' phonological awareness (PA) and alphabet knowledge (AK) skills are two of the strongest predictors of future reading. Despite evidence that providing at-risk preschoolers with timely emergent literacy interventions can prevent academic difficulties, there is a scarcity of research focusing on Latinx preschoolers who are dual language learners. Despite evidence of benefits of providing Latinxs with Spanish emergent literacy instruction, few studies include preschoolers. This study examined the effects of a supplemental Spanish PA and AK intervention on the dual emergent literacy skills of at-risk Latinx preschoolers. Method A multiple probe design across four units of instruction evaluated the effects of a Spanish supplemental emergent literacy intervention that explicitly facilitated generalizations to English. Four Latinx preschoolers with limited emergent literacy skills in Spanish and English participated in this study. Bilingual researchers delivered scripted lessons targeting PA and AK skills in individual or small groups for 12–17 weeks. Results Children made large gains as each PA skill was introduced into intervention and generalized the PA skills they learned from Spanish to English. They also improved their English initial sound identification skills, a phonemic awareness task, when instruction was delivered in Spanish but with English words. Children made small to moderate gains in their Spanish letter naming and letter–sound correspondence skills and in generalizing this knowledge to English. Conclusion These findings provide preliminary evidence Latinx preschoolers who are dual language learners benefit from emergent literacy instruction that promotes their bilingual and biliterate development.

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