scholarly journals Protocols for 16S rDNA Array Analyses of Microbial Communities by Sequence-Specific Labeling of DNA Probes

2003 ◽  
Vol 3 ◽  
pp. 578-584 ◽  
Author(s):  
Knut Rudi ◽  
Janneke Treimo ◽  
Hilde Nissen ◽  
Gerd Vegarud
Keyword(s):  
Microbial Communities ◽  
Pcr Amplification ◽  
Dna Probes ◽  
Dna Array ◽  
Gene Encoding ◽  
Variable Regions ◽  
Conserved Regions ◽  
Rdna Array ◽  
Array Hybridization ◽  
Array Analyses

Analyses of complex microbial communities are becoming increasingly important. Bottlenecks in these analyses, however, are the tools to actually describe the biodiversity. Novel protocols for DNA array-based analyses of microbial communities are presented. In these protocols, the specificity obtained by sequence-specific labeling of DNA probes is combined with the possibility of detecting several different probes simultaneously by DNA array hybridization. The gene encoding 16S ribosomal RNA was chosen as the target in these analyses. This gene contains both universally conserved regions and regions with relatively high variability. The universally conserved regions are used for PCR amplification primers, while the variable regions are used for the specific probes. Protocols are presented for DNA purification, probe construction, probe labeling, and DNA array hybridizations.

scholarly journals CaptureSeq: Hybridization-Based Enrichment of cpn60 Gene Fragments Reveals the Community Structures of Synthetic and Natural Microbial Ecosystems

2021 ◽  
Vol 9 (4) ◽  
pp. 816
Author(s):  
Matthew G. Links ◽  
Tim J. Dumonceaux ◽  
E. Luke McCarthy ◽  
Sean M. Hemmingsen ◽  
Edward Topp ◽  
...  
Keyword(s):  
Microbial Community ◽  
Microbial Communities ◽  
Dna Sequences ◽  
Pcr Amplification ◽  
Shotgun Sequencing ◽  
Natural Ecosystems ◽  
Gene Markers ◽  
Microbial Profile ◽  
Microbial Ecosystems ◽  
Pcr Targeting

Background. The molecular profiling of complex microbial communities has become the basis for examining the relationship between the microbiome composition, structure and metabolic functions of those communities. Microbial community structure can be partially assessed with “universal” PCR targeting taxonomic or functional gene markers. Increasingly, shotgun metagenomic DNA sequencing is providing more quantitative insight into microbiomes. However, both amplicon-based and shotgun sequencing approaches have shortcomings that limit the ability to study microbiome dynamics. Methods. We present a novel, amplicon-free, hybridization-based method (CaptureSeq) for profiling complex microbial communities using probes based on the chaperonin-60 gene. Molecular profiles of a commercially available synthetic microbial community standard were compared using CaptureSeq, whole metagenome sequencing, and 16S universal target amplification. Profiles were also generated for natural ecosystems including antibiotic-amended soils, manure storage tanks, and an agricultural reservoir. Results. The CaptureSeq method generated a microbial profile that encompassed all of the bacteria and eukaryotes in the panel with greater reproducibility and more accurate representation of high G/C content microorganisms compared to 16S amplification. In the natural ecosystems, CaptureSeq provided a much greater depth of coverage and sensitivity of detection compared to shotgun sequencing without prior selection. The resulting community profiles provided quantitatively reliable information about all three domains of life (Bacteria, Archaea, and Eukarya) in the different ecosystems. The applications of CaptureSeq will facilitate accurate studies of host-microbiome interactions for environmental, crop, animal and human health. Conclusions: cpn60-based hybridization enriched for taxonomically informative DNA sequences from complex mixtures. In synthetic and natural microbial ecosystems, CaptureSeq provided sequences from prokaryotes and eukaryotes simultaneously, with quantitatively reliable read abundances. CaptureSeq provides an alternative to PCR amplification of taxonomic markers with deep community coverage while minimizing amplification biases.

scholarly journals PacBio Amplicon Sequencing Method To Measure Pilin Antigenic Variation Frequencies of Neisseria gonorrhoeae

mSphere ◽  
2019 ◽  
Vol 4 (5) ◽  
Author(s):  
Egon A. Ozer ◽  
Lauren L. Prister ◽  
Shaohui Yin ◽  
Billy H. Ward ◽  
Stanimir Ivanov ◽  
...  
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Antigenic Variation ◽  
Amplicon Sequencing ◽  
Common Mechanism ◽  
Macrophage Infection ◽  
Gene Encoding ◽  
Transmitted Infection ◽  
Variable Regions ◽  
Content Type ◽  
Strain Fa1090

ABSTRACT Gene diversification is a common mechanism pathogens use to alter surface structures to aid in immune avoidance. Neisseria gonorrhoeae uses a gene conversion-based diversification system to alter the primary sequence of the gene encoding the major subunit of the pilus, pilE. Antigenic variation occurs when one of the nonexpressed 19 silent copies donates part of its DNA sequence to pilE. We have developed a method using Pacific Biosciences (PacBio) amplicon sequencing and custom software to determine pilin antigenic variation frequencies. The program analyzes 37 variable regions across the strain FA1090 1-81-S2 pilE gene and can be modified to determine sequence variation from other starting pilE sequences or other diversity generation systems. Using this method, we measured pilin antigenic variation frequencies for various derivatives of strain FA1090 and showed we can also analyze pilin antigenic variation frequencies during macrophage infection. IMPORTANCE Diversity generation systems are used by many unicellular organism to provide subpopulations of cell with different properties that are available when needed. We have developed a method using the PacBio DNA sequencing technology and a custom computer program to analyze the pilin antigenic variation system of the organism that is the sole cause of the sexually transmitted infection, gonorrhea.

scholarly journals Plasmid-Encoded Metallo-β-Lactamase (IMP-6) Conferring Resistance to Carbapenems, Especially Meropenem

2001 ◽  
Vol 45 (5) ◽  
pp. 1343-1348 ◽  
Author(s):  
Hisakazu Yano ◽  
Akio Kuga ◽  
Ryoichi Okamoto ◽  
Hidero Kitasato ◽  
Toshimitsu Kobayashi ◽  
...  
Keyword(s):  
Point Mutation ◽  
Antimicrobial Agents ◽  
Pcr Amplification ◽  
Penicillin G ◽  
Gene Encoding ◽  
Mature Enzyme ◽  
Dna Fragment ◽  
Lactamase Gene ◽  
Reduced Activity ◽  
Northern Japan

ABSTRACT In 1996, Serratia marcescens KU3838 was isolated from the urine of a patient with a urinary tract infection at a hospital in northern Japan and was found to contain the plasmid pKU501. Previously, we determined that pKU501 carries bla IMP and the genes for TEM-1-type β-lactamases as well as producing both types of β-lactamases (H. Yano, A. Kuga, K. Irinoda, R. Okamoto, T. Kobayashi, and M. Inoue, J. Antibiot. 52:1135–1139, 1999). pKU502 is a recombinant plasmid that contains a 1.5-kb DNA fragment, including the metallo-β-lactamase gene, and is obtained by PCR amplification of pKU501. The sequence of the metallo-β-lactamase gene in pKU502 was determined and revealed that this metallo-β-lactamase gene differed from the gene encoding IMP-1 by one point mutation, leading to one amino acid substitution: 640-A in the base sequence of the IMP-1 gene was replaced by G, and Ser-196 was replaced by Gly in the mature enzyme. This enzyme was designated IMP-6. The strains that produced IMP-6 were resistant to carbapenems. The MICs of panipenem and especially meropenem were higher than the MIC of imipenem for these strains. The k cat/Km value of IMP-6 was about sevenfold higher against meropenem than against imipenem, although the MIC of meropenem for KU1917, which produced IMP-1, was lower than that of imipenem, and the MIC of panipenem was equal to that of imipenem. These results support the hypothesis that IMP-6 has extended substrate profiles against carbapenems. However, the activity of IMP-6 was very low against penicillin G and piperacillin. These results suggest that IMP-6 acquired high activity against carbapenems, especially meropenem, via the point mutation but in the process lost activity against penicillins. Although IMP-6 has reduced activity against penicillins due to this point mutation, pKU501 confers resistance to a variety of antimicrobial agents because it also produces TEM-1-type enzyme.

scholarly journals PCR amplification of antibody variable regions using primers that anneal to constant regions

1994 ◽  
Vol 22 (9) ◽  
pp. 1768-1769 ◽  
Author(s):  
Marica Sassano ◽  
Monica Repetto ◽  
Giovanni Cassani ◽  
Angelo Corti
Keyword(s):  
Pcr Amplification ◽  
Variable Regions

scholarly journals Autonomous assembly of synthetic oligonucleotides built from an expanded DNA alphabet. Total synthesis of a gene encoding kanamycin resistance

2014 ◽  
Vol 10 ◽  
pp. 2348-2360 ◽  
Author(s):  
Kristen K Merritt ◽  
Kevin M Bradley ◽  
Daniel Hutter ◽  
Mariko F Matsuura ◽  
Diane J Rowold ◽  
...  
Keyword(s):  
Solid Phase ◽  
Pcr Amplification ◽  
Kanamycin Resistance ◽  
Dna Fragments ◽  
Gene Encoding ◽  
Synthetic Dna ◽  
Information Density ◽  
Letter Alphabet ◽  
New Strategy ◽  
Autonomous Assembly

Background: Many synthetic biologists seek to increase the degree of autonomy in the assembly of long DNA (L-DNA) constructs from short synthetic DNA fragments, which are today quite inexpensive because of automated solid-phase synthesis. However, the low information density of DNA built from just four nucleotide “letters”, the presence of strong (G:C) and weak (A:T) nucleobase pairs, the non-canonical folded structures that compete with Watson–Crick pairing, and other features intrinsic to natural DNA, generally prevent the autonomous assembly of short single-stranded oligonucleotides greater than a dozen or so. Results: We describe a new strategy to autonomously assemble L-DNA constructs from fragments of synthetic single-stranded DNA. This strategy uses an artificially expanded genetic information system (AEGIS) that adds nucleotides to the four (G, A, C, and T) found in standard DNA by shuffling hydrogen-bonding units on the nucleobases, all while retaining the overall Watson–Crick base-pairing geometry. The added information density allows larger numbers of synthetic fragments to self-assemble without off-target hybridization, hairpin formation, and non-canonical folding interactions. The AEGIS pairs are then converted into standard pairs to produce a fully natural L-DNA product. Here, we report the autonomous assembly of a gene encoding kanamycin resistance using this strategy. Synthetic fragments were built from a six-letter alphabet having two AEGIS components, 5-methyl-2’-deoxyisocytidine and 2’-deoxyisoguanosine (respectively S and B), at their overlapping ends. Gaps in the overlapped assembly were then filled in using DNA polymerases, and the nicks were sealed by ligase. The S:B pairs in the ligated construct were then converted to T:A pairs during PCR amplification. When cloned into a plasmid, the product was shown to make Escherichia coli resistant to kanamycin. A parallel study that attempted to assemble similarly sized genes with optimally designed standard nucleotides lacking AEGIS components gave successful assemblies of up to 16 fragments, but generally failed when larger autonomous assemblies were attempted. Conclusion: AEGIS nucleotides, by increasing the information density of DNA, allow larger numbers of DNA fragments to autonomously self-assemble into large DNA constructs. This technology can therefore increase the size of DNA constructs that might be used in synthetic biology.

scholarly journals ISOLASI FRAGMEN GEN PENYANDI PUTRESIN N-METILTRANSFERASE DAN QUINOLINAT FOSFORIBOSILTRANSFERASE ASAL TEMBAKAU LOKAL TEMANGGUNG (Nicotiana tabacum)

2020 ◽  
Vol 17 (3) ◽  
pp. 109 ◽  
Author(s):  
SESANTI BASUKI ◽  
NURHAJATI AA MATTJIK ◽  
SUWARSO SUWARSO ◽  
DESTA WIRNAS ◽  
SUDARSONO SUDARSONO
Keyword(s):  
Dna Sequences ◽  
Pcr Amplification ◽  
Data Bank ◽  
Degenerate Primer ◽  
Gene Encoding ◽  
Methyl Transferase ◽  
Dna Database ◽  
Genes Encoding ◽  
Tobacco Cultivar ◽  
Dna Template

<p>ABSTRAK</p><p>Upaya untuk menurunkan kandungan nikotin merupakan salah satuprioritas utama penelitian tembakau. Nikotin adalah senyawa alkaloidutama berpotensi dikonversi menjadi senyawa nor-nikotin yang bersifatkarsinogen. Gen PMT sebagai penyandi enzim putresin n-metiltransferase(PMT) dan gen QPT - penyandi enzim quinolinat fosforibosiltransferase(QPT) merupakan dua gen kunci yang berperan penting pada proses bio-sintesis nikotin. Penelitian ini bertujuan untuk mengisolasi potongan genPMT dan QPT asal tembakau lokal Indonesia, mengkarakterisasi danmenganalisis runutan DNA-nya. Tahapan penelitian dimulai dengan me-rancang primer degenerate berdasarkan informasi yang ada di pangkalandata Bank Gen NCBI (National Centre for Biotechnology Information),mengamplifikasi PCR menggunakan templat DNA genomik tembakaulokal cv. Sindoro1, mengklon potongan DNA hasil PCR dan menentukanrunutan DNA-nya. Hasil penelitian menunjukkan dari dua belas pasangprimer degenerate yang dirancang, hanya dua pasang primer yang meng-hasilkan potongan DNA hasil amplifikasi PCR, yaitu pasangan primerPMt-7 (F &amp; R) untuk gen PMT dan primer QPt-3 (F &amp; R) untuk gen QPT.Setelah dilakukan penentuan runutan DNA-nya, amplikon yang didapatdari hasil PCR dengan pasangan primer PMt-7 sebesar 1418 bp, sedangkanuntuk primer QPt-3 sebesar 205 bp. Runutan DNA gen PMT dan gen QPTasal tembakau lokal cv. Sindoro1 mempunyai tingkat kesamaan yang ting-gi dengan gen PMT dan gen QPT asal tembakau lainnya yang ada dipangkalan data Bank Gen NCBI.</p><p>Kata kunci : Gen PMT, gen QPT, lintasan biosintesis nikotin, perunutanDNA, amplifikasi PCR, primer degenerate</p><p>ABSTRACT</p><p>Isolation of Genes encoding Putrescine N-Methyl-transferase and Quinolinat Phosphoribosyl transferasederived from Temanggung Tobacco Cultivar (Nicotianatabacum)</p><p>Reduction of nicotine content is one of the major objective intobacco research. Nicotine is the main alcaloid compound that potentiallycould be converted into a carcinogenic compound (nor-nicotine). The PMTgene encoding putrescine N-methyl transferase (PMT) and the QPT gene -encoding quinolinate phosphoribosyl transferase (QPT) are the two keyenzymes involved in nicotine biosynthesis. The objectives of this researchwere to isolate PMT and QPT gene fragments originated from Indonesianlocal tobacco, to characterize, and to analyze their DNA sequences. Theresearch activities included: degenerate primer design based oninformation available in the GenBank DNA Database NCBI (NationalCentre for Biotechnology Information), PCR amplification usingdegenerate primer and genomic DNA template of a local tobacco cv.Sindoro1, clone the PCR amplified products, and determine their DNAnucleotide sequences. Results of the experiment indicated that from 12degenerate primer pairs synthesized, only two were able to yield positivePCR amplified products. These primer pairs were PMt-7 (F &amp; R primers)for PMT and QPt-3 (F &amp; R primers) for QPT. After DNA sequencing, theamplified DNA product amplified using PMt-7 degenerate primer pairswere 1418 bp, while that using QPt-3 primer pairs were only 205 bp.Nucleotide sequences of PMT or QPT gene fragments originated fromlocal tobacco cv. Sindoro1 showed a high nucleotide sequences identity ascompared to that of the respective genes from other tobacco species thatwere available in the GenBank DNA Database NCBI.</p><p>Key words: PMT gene, QPT gene, nicotine biosynthetic pathways, DNAsequencing, PCR amplification, degenerate primer</p>

Molecular characterization and structure of intestinal micro flora for postmortem interval estimation in SD rats

2019 ◽  
Author(s):  
Huan Li ◽  
Lu Yuan ◽  
Ruina Liu ◽  
Siruo Zhang ◽  
E Yang ◽  
...  
Keyword(s):  
Bacterial Community ◽  
High Throughput Sequencing ◽  
Interval Estimation ◽  
Pcr Amplification ◽  
Postmortem Interval ◽  
The Body ◽  
Rrna Gene ◽  
Variable Regions ◽  
Time Points ◽  
Almost All

Abstract Background The human rectum flora consists of a huge variety of bacteria and the association between individuals and their rectum bacterial community begins presently after birth and continues the whole lifetime. Once the body dies, the inherent microbes begin to break down from the inside and play a key role thereafter. Results The aim of this study was to investigate the probable shift of the rectum flora at different time intervals up to 15 days after death and to characterize the contribution for of this shift to estimate the time of death. The rectum of rats was wiped with a sterile cotton swab and the samples were proceeded for DNA extraction, PCR amplification of the 16S rRNA gene with the V3+V4 variable regions, and high throughput sequencing carried out on IonS5TMXL platform. The results were analyzed for intra-group and inter-group diversity, similarity and difference at different time points. At phylum level, Proteobacteria and Firmicutes showed major shifts, checked at 11 different intervals and emerged in the most of postmortem intervals. At the genus level, Enterococcus appeared in all groups except alive samples, Lactobacillus and Proteus appeared in most time points, and the latter showed an increasing trend after 3 days postmortem samples. At the species level, Enterococcus_faecalis and Proteus_mirabilis existed in most postmortem intervals, and the former had a downward trend after day 5 postmortem, while the latter had an upward trend. Corynebacterium_amycolatum , Entero_isolate_group_2 , Bacteroides_uniformis , Enterococcus_faecalis , Streptococcus_gallolyticus_subsp_macedonics , Clostridium_sporogenes were more abundant in 0-hour, day 1, 3, 5, 7, 13 postmortem intervals, respectively, while Proteus_mirabilis and Vagococcus_lutrae were abundant in day 15 postmortem. In addition, functional capacity analysis of Membrane_Transport, Amino_Acid_Metabolism, Nucleotide_Metabolism and Energy_Metabolism showed significant differences between alive and almost all other time points after death ( P <0.05). Conclusions All in all, bacteria at different levels (phylum, genera, species) showed different characteristic during the process of decomposition and possessed entirely different relative abundance and the structure of bacterial community in each time point shifted obviously, which suggested that the specific bacteria might imply the specific postmortem interval during decomposition.

scholarly journals Characterization of Flagellotropic, Chi-Like Salmonella Phages Isolated from Thai Poultry Farms

Viruses ◽  
2019 ◽  
Vol 11 (6) ◽  
pp. 520 ◽  
Author(s):  
Preeda Phothaworn ◽  
Matthew Dunne ◽  
Rattaya Supokaivanich ◽  
Catherine Ong ◽  
Jiali Lim ◽  
...  
Keyword(s):  
Salmonella Enterica Serovar Typhimurium ◽  
Sequence Similarity ◽  
Phage Type ◽  
Pcr Amplification ◽  
Phylogenomic Analysis ◽  
Monophyletic Clade ◽  
Gene Encoding ◽  
Poultry Farms ◽  
Serovar Typhimurium

Despite a wealth of knowledge on Salmonella phages worldwide, little is known about poultry-associated Salmonella phages from Thailand. Here, we isolated 108 phages from Thai poultry farms that infect Salmonella enterica serovar Typhimurium. Phages STm101 and STm118 were identified as temperate Siphoviridae phages. Genome sequencing and analyses revealed these phages share approximately 96% nucleotide sequence similarity to phage SPN19, a member of the Chi-like virus genus. PCR amplification of the gene encoding capsid protein E of the Chi-like phage was positive for 50% of phage isolates, suggesting a predominance of this phage type among the sampled poultry farms. In addition to the flagella, two phages required the lipopolysaccharide to infect and lyse Salmonella. Furthermore, phylogenomic analysis demonstrated that phages STm101 and STm118 formed a monophyletic clade with phages isolated from Western countries, but not from closer isolated phages from Korea. However, further investigation and more phage isolates are required to investigate possible causes for this geographic distribution.

scholarly journals Comparison of Extracellular Enzyme Activities and Community Composition of Attached and Free-Living Bacteria in Porous Medium Columns

2002 ◽  
Vol 68 (4) ◽  
pp. 1569-1575 ◽  
Author(s):  
R. Michael Lehman ◽  
Seán P. O'Connell
Keyword(s):  
Porous Medium ◽  
Microbial Communities ◽  
Enzyme Activities ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Solid Phase ◽  
Pcr Amplification ◽  
Extracellular Enzyme ◽  
Free Living ◽  
Extracellular Enzyme Activities ◽  
Community Function

ABSTRACT Free-living and surface-associated microbial communities in sand-packed columns perfused with groundwater were compared by examination of compositional and functional characteristics. The composition of the microbial communities was assessed by bulk DNA extraction, PCR amplification of 16S ribosomal DNA fragments, separation of these fragments by denaturing gradient gel electrophoresis, and sequence analysis. Community function was assessed by measurement of β-glucosidase and aminopeptidase extracellular enzyme activities. Free-living populations in the aqueous phase exhibited a greater diversity of phylotypes than populations associated with the solid phase. The attached bacterial community displayed significantly greater β-glucosidase and aminopeptidase enzyme activities per volume of porous medium than those of the free-living community. On a per-cell basis, the attached community had a significantly higher cell-specific aminopeptidase enzyme activity (1.07 × 10−7 nmol cell−1 h−1) than the free-living community (5.02 × 10−8 nmol cell−1 h−1). Conversely, the free-living community had a significantly higher cell-specific β-glucosidase activity (1.92 × 10−6 nmol cell−1 h−1) than the surface-associated community (6.08 × 10−7 nmol cell−1 h−1). The compositional and functional differences observed between these two communities may reflect different roles for these distinct but interacting communities in the decomposition of natural organic matter or biodegradation of xenobiotics in aquifers.

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