scholarly journals Sequence analysis of 12S rRNA and 16S rRNA mitochondrial genes in Iranian Afshari sheep

2019 ◽  
Vol 8 (1) ◽  
Author(s):  
Mohammad Mahmoodi ◽  
Kian Pahlevan Afshari ◽  
Hamid Reza Seyedabadi ◽  
Mehran Aboozari
Keyword(s):  
Sequence Analysis ◽  
16S Rrna ◽  
Pcr Amplification ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Conservation Strategies ◽  
12S Rrna ◽  
Rrna Gene ◽  
Salting Out ◽  
The Rich

Phylogenetic relationships and genetic variation in Iranian Afshari sheep breed were analyzed using 12S rRNA and 16S rRNA gene sequences. The genomic DNA was isolated by salting out method and amplified 12S rRNA and 16S rRNA genes using PCR method. PCR amplification of 12S and 16S rRNA generated PCR amplicons at 859 and 1053 bp lengths, respectively. Sequence analysis was performed using BioEdit software. Phylogenetic tree was constructed using MEGA software. Phylogenetic analysis of haplotype in the combination with the sheep from GenBank showed that Iranian Afshari sheep made a close to the Australian sheep cluster. This study was found informative for establishing relationships between breeds from different parts of the world. This study may facilitate the future researchers and breeders for better understanding the genetic interactions and breed differentiation for devising future breeding and conservation strategies to preserve the rich animal genetic reservoir of the country.

scholarly journals Oligonucleotide Microarray for 16S rRNA Gene-Based Detection of All Recognized Lineages of Sulfate-Reducing Prokaryotes in the Environment

2002 ◽  
Vol 68 (10) ◽  
pp. 5064-5081 ◽  
Author(s):  
Alexander Loy ◽  
Angelika Lehner ◽  
Natuschka Lee ◽  
Justyna Adamczyk ◽  
Harald Meier ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Pcr Amplification ◽  
Oligonucleotide Microarray ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Clinical Samples ◽  
Rrna Gene ◽  
Sulfate Reducing ◽  
Specific Pcr

ABSTRACT For cultivation-independent detection of sulfate-reducing prokaryotes (SRPs) an oligonucleotide microarray consisting of 132 16S rRNA gene-targeted oligonucleotide probes (18-mers) having hierarchical and parallel (identical) specificity for the detection of all known lineages of sulfate-reducing prokaryotes (SRP-PhyloChip) was designed and subsequently evaluated with 41 suitable pure cultures of SRPs. The applicability of SRP-PhyloChip for diversity screening of SRPs in environmental and clinical samples was tested by using samples from periodontal tooth pockets and from the chemocline of a hypersaline cyanobacterial mat from Solar Lake (Sinai, Egypt). Consistent with previous studies, SRP-PhyloChip indicated the occurrence of Desulfomicrobium spp. in the tooth pockets and the presence of Desulfonema- and Desulfomonile-like SRPs (together with other SRPs) in the chemocline of the mat. The SRP-PhyloChip results were confirmed by several DNA microarray-independent techniques, including specific PCR amplification, cloning, and sequencing of SRP 16S rRNA genes and the genes encoding the dissimilatory (bi)sulfite reductase (dsrAB).

scholarly journals Multilocus sequence analysis of Ensifer and related taxa

2007 ◽  
Vol 57 (3) ◽  
pp. 489-503 ◽  
Author(s):  
Miet Martens ◽  
Manuel Delaere ◽  
Renata Coopman ◽  
Paul De Vos ◽  
Monique Gillis ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
16S Rrna ◽  
Housekeeping Genes ◽  
16S Rrna Genes ◽  
New Combination ◽  
Rrna Genes ◽  
Multilocus Sequence Analysis ◽  
Bootstrap Support ◽  
Rrna Gene ◽  
Separate Species

Multilocus sequence analysis (MLSA) was performed on representatives of Ensifer (including species previously assigned to the genus Sinorhizobium) and related taxa. Neighbour-joining (NJ), maximum-parsimony (MP) and maximum-likelihood (ML) phylogenies of dnaK, gltA, glnA, recA, thrC and 16S rRNA genes were compared. The data confirm that the potential for discrimination of Ensifer species is greater using MLSA of housekeeping genes than 16S rRNA genes. In incongruence-length difference tests, the 16S rRNA gene was found to be significantly incongruent with the other genes, indicating that this gene should not be used as a single indicator of relatedness in this group. Significant congruence was detected for dnaK, glnA and thrC. Analyses of concatenated sequences of dnaK, glnA and thrC genes yielded very similar NJ, MP and ML trees, with high bootstrap support. In addition, analysis of a concatenation of all six genes essentially produced the same result, levelling out potentially conflicting phylogenetic signals. This new evidence supports the proposal to unite Ensifer and Sinorhizobium in a single genus. Support for an alternative solution preserving the two genera is less strong. In view of the opinions expressed by the Judicial Commission, the name of the genus should be Ensifer, as proposed by Young [Young, J. M. (2003). Int J Syst Evol Microbiol 53, 2107–2110]. Data obtained previously and these new data indicate that Ensifer adhaerens and ‘Sinorhizobium morelense’ are not heterotypic synonyms, but represent separate species. However, transfer to the genus Ensifer is not possible at present because the species name is the subject of a pending Request for an Opinion, which would affect whether a novel species in the genus Ensifer or a new combination based on a basonym would be created.

scholarly journals The Influence of DNA Extraction Procedure and Primer Set on the Bacterial Community Analysis by Pyrosequencing of Barcoded 16S rRNA Gene Amplicons

2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Ingo C. Starke ◽  
Wilfried Vahjen ◽  
Robert Pieper ◽  
Jürgen Zentek
Keyword(s):  
Sequence Analysis ◽  
16S Rrna ◽  
Dna Extraction ◽  
16S Rrna Genes ◽  
Read Length ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Variable Regions ◽  
Extraction Procedures ◽  
Primer Sets

In this study, the effect of different DNA extraction procedures and primer sets on pyrosequencing results regarding the composition of bacterial communities in the ileum of piglets was investigated. Ileal chyme from piglets fed a diet containing different amounts of zinc oxide was used to evaluate a pyrosequencing study with barcoded 16S rRNA PCR products. Two DNA extraction methods (bead beating versus silica gel columns) and two primer sets targeting variable regions of bacterial 16S rRNA genes (8f-534r versus 968f-1401r) were considered. The SEED viewer software of the MG-RAST server was used for automated sequence analysis. A total of 5.2×105 sequences were used for analysis after processing for read length (150 bp), minimum sequence occurrence (5), and exclusion of eukaryotic and unclassified/uncultured sequences. DNA extraction procedures and primer sets differed significantly in total sequence yield. The distribution of bacterial order and main bacterial genera was influenced significantly by both parameters. However, this study has shown that the results of pyrosequencing studies using barcoded PCR amplicons of bacterial 16S rRNA genes depend on DNA extraction and primer choice, as well as on the manner of downstream sequence analysis.

scholarly journals Selective and Sensitive Method for PCR Amplification of Escherichia coli 16S rRNA Genes in Soil

2000 ◽  
Vol 66 (2) ◽  
pp. 844-849 ◽  
Author(s):  
G. Sabat ◽  
P. Rose ◽  
W. J. Hickey ◽  
J. M. Harkin
Keyword(s):  
Escherichia Coli ◽  
16S Rrna ◽  
Pcr Amplification ◽  
Pcr Primers ◽  
Enteric Bacteria ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Selective Amplification ◽  
E Coli

ABSTRACT A set of PCR primers targeting 16S rRNA gene sequences was designed, and PCR parameters were optimized to develop a robust and reliable protocol for selective amplification of Escherichia coli 16S rRNA genes. The method was capable of discriminatingE. coli from other enteric bacteria, including its closest relative, Shigella. Selective amplification of E. coli occurred only when the annealing temperature in the PCR was elevated to 72°C, which is 10°C higher than the optimum for the primers. Sensitivity was retained by modifying the length of steps in the PCR, by increasing the number of cycles, and most importantly by optimizing the MgCl2 concentration. The PCR protocol developed can be completed in less then 2 h and, by using Southern hybridization, has a detection limit of ca. 10 genomic equivalents per reaction. The method was demonstrated to be effective for detectingE. coli DNA in heterogeneous DNA samples, such as those extracted from soil.

scholarly journals Target enrichment from a DNA mixture by oligoribonucleotide interference-PCR (ORNi-PCR)

2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Toshitsugu Fujita ◽  
Daisuke Motooka ◽  
Hodaka Fujii
Keyword(s):  
16S Rrna ◽  
Genome Editing ◽  
Dna Sequences ◽  
Pcr Amplification ◽  
Dna Amplification ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Detailed Assessment ◽  
Dna Mixture

Abstract Oligoribonucleotide (ORN) interference-PCR (ORNi-PCR) is a method that suppresses PCR amplification of target DNA in an ORN-specific manner. In this study, we examined whether ORNi-PCR can be used to enrich desirable DNA sequences from a DNA mixture by suppressing undesirable DNA amplification. ORNi-PCR enriched edited DNA sequences from a mixture of genomic DNA subjected to genome editing. ORNi-PCR enabled more efficient analysis of the types of insertion/deletion mutations introduced by genome editing. In addition, ORNi-PCR reduced the detection of 16S ribosomal RNA (16S rRNA) genes in 16S rRNA gene-based microbiome profiling, which might permit a more detailed assessment of populations of other 16S rRNA genes. Enrichment of desirable DNA sequences by ORNi-PCR may be useful in molecular biology, medical diagnosis, and other fields.

scholarly journals Molecular characterization of Mycobacterium intracellulare-related strains based on the sequence analysis of hsp65, internal transcribed spacer and 16S rRNA genes

2010 ◽  
Vol 59 (9) ◽  
pp. 1037-1043 ◽  
Author(s):  
Joo-Hee Park ◽  
Tae-Sun Shim ◽  
Seung-Ae Lee ◽  
Hyungki Lee ◽  
In-Kyung Lee ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
16S Rrna ◽  
Internal Transcribed Spacer ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Mycobacterium Intracellulare ◽  
Epidemiological Features ◽  
The 16S Rrna Gene

We investigated the molecular epidemiological features of 94 Mycobacterium intracellulare-related strains, isolated from Korean patients, using sequence analysis targeting 3 independent chronometer molecules, hsp65, the internal transcribed spacer 1 region and the 16S rRNA gene. By collective consideration of these three gene-based approaches, the 94 strains were divided into 5 groups (INT1, INT2, INT3, INT4 and INT5). The frequencies of genotype INT1, 2, 3, 4 and 5 in the 94 isolates were 57.4 % (54), 27.7 % (26), 6.4 % (6), 5.3 % (5) and 3.2 % (3), respectively. When correlations between genotypes and clinical parameters (age, sex, radiological type and the presence of a cavity) were analysed in 78 patients with non-tuberculous mycobacteria pulmonary diseases, no relationships were observed with respect to age, sex and radiological type, but genotype and the presence of a cavity tended to be related (P=0.051).

scholarly journals Identification and classification of the genus Bacteroides by multilocus sequence analysis

Microbiology ◽  
2011 ◽  
Vol 157 (12) ◽  
pp. 3388-3397 ◽  
Author(s):  
Mitsuo Sakamoto ◽  
Moriya Ohkuma
Keyword(s):  
Sequence Analysis ◽  
16S Rrna ◽  
Sequence Similarity ◽  
Single Gene ◽  
Housekeeping Genes ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Multilocus Sequence Analysis ◽  
Rrna Gene

Multilocus sequence analysis (MLSA) was performed on representative species of the genus Bacteroides. Internal fragments of the genes selected, dnaJ, gyrB, hsp60, recA, rpoB and 16S rRNA, were amplified by direct PCR and then sequenced from 38 Bacteroides strains representing 35 species. Neighbour-joining (NJ), maximum-likelihood (ML) and maximum-parsimony (MP) phylogenies of the individual genes were compared. The data confirm that the potential for discrimination of Bacteroides species is greater using MLSA of housekeeping genes than 16S rRNA genes. Among the housekeeping genes analysed, gyrB was the most informative, followed by dnaJ. Analyses of concatenated sequences (4816 bp) of all six genes revealed robust phylogenetic relationships among different Bacteroides species when compared with the single-gene trees. The NJ, ML and MP trees were very similar, and almost fully resolved relationships of Bacteroides species were obtained, to our knowledge for the first time. In addition, analysis of a concatenation (2457 bp) of the dnaJ, gyrB and hsp60 genes produced essentially the same result. Ten distinct clades were recognized using the SplitsTree4 program. For the genus Bacteroides, we can define species as a group of strains that share at least 97.5 % gene sequence similarity based on the fragments of five protein-coding housekeeping genes and the 16S rRNA gene. This study demonstrates that MLSA of housekeeping genes is a valuable alternative technique for the identification and classification of species of the genus Bacteroides.

scholarly journals Screening of a Fosmid Library of Marine Environmental Genomic DNA Fragments Reveals Four Clones Related to Members of the OrderPlanctomycetales

1998 ◽  
Vol 64 (8) ◽  
pp. 3075-3078 ◽  
Author(s):  
Kevin L. Vergin ◽  
Ena Urbach ◽  
Jeffery L. Stein ◽  
Edward F. DeLong ◽  
Brian D. Lanoil ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
16S Rrna ◽  
Genomic Dna ◽  
Pcr Amplification ◽  
Fosmid Library ◽  
Full Length ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Bacterial Dna ◽  
Marine Environmental

ABSTRACT A fosmid library with inserts containing approximately 40 kb of marine bacterial DNA (J. L. Stein, T. L. Marsh, K. Y. Wu, H. Shizuya, and E. F. DeLong, J. Bacteriol. 178:591–599, 1996) yielded four clones with 16S rRNA genes from the orderPlanctomycetales. Three of the clones belong to thePirellula group and one clone belongs to thePlanctomyces group, based on phylogenetic and signature nucleotide analyses of full-length 16S rRNA genes. Sequence analysis of the ends of the genes revealed a consistent mismatch in a widely used bacterium-specific 16S rRNA PCR amplification priming site (27F), which has also been reported in some thermophiles and spirochetes.

scholarly journals Flow-Cytometric Cell Sorting and Subsequent Molecular Analyses for Culture-Independent Identification of Bacterioplankton Involved in Dimethylsulfoniopropionate Transformations

2005 ◽  
Vol 71 (3) ◽  
pp. 1405-1416 ◽  
Author(s):  
Xiaozhen Mou ◽  
Mary Ann Moran ◽  
Ramunas Stepanauskas ◽  
José M. González ◽  
Robert E. Hodson
Keyword(s):  
16S Rrna ◽  
Cell Sorting ◽  
Marine Ecosystem ◽  
Pcr Amplification ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Coastal Marine ◽  
Four Seasons ◽  
Culture Independent

ABSTRACT Marine bacterioplankton transform dimethylsulfoniopropionate (DMSP) into the biogeochemically important and climatically active gas dimethylsulfide. In order to identify specific bacterial taxa mediating DMSP processing in a natural marine ecosystem, we amended water samples from a southeastern U.S. salt marsh with 20 μM DMSP and tracked community shifts with flow cytometry (FCM) coupled to 16S rRNA gene analyses. In two out of four seasons studied, DMSP amendments induced the formation of distinct bacterioplankton populations with elevated nucleic acid (NA) content within 24 h, indicative of cells actively utilizing DMSP. The 16S rRNA genes of the cells with and without elevated NA content were analyzed following cell sorting and PCR amplification with sequencing and terminal restriction fragment length polymorphism approaches. Compared to cells in the control FCM populations, bacteria with elevated NA content in the presence of DMSP were relatively enriched in taxa related to Loktanella, Oceanicola, and Sulfitobacter (Roseobacter lineage, α-Proteobacteria); Caulobacter (α-Proteobacteria); and Brachymonas and Xenophilus (β-Proteobacteria) in the May-02 sample and to Ketogulonicigenium (Roseobacter lineage, α-Proteobacteria) and novel γ-Proteobacteria in the Sept-02 sample. Our study suggests that diverse bacterioplankton participate in the metabolism of DMSP in coastal marine systems and that their relative importance varies temporally.

scholarly journals Identification and comparison of three carp fishes based on mitochondrial 16S rRNA gene

2017 ◽  
Vol 26 (2) ◽  
pp. 167-174
Author(s):  
Hawa Jahan ◽  
Maria Akter ◽  
Rowshan Ara Begum ◽  
Reza Md Shahjahan
Keyword(s):  
16S Rrna ◽  
Developmental Stages ◽  
Pcr Amplification ◽  
Morphological Features ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Universal Primers ◽  
Rrna Gene ◽  
Multiple Sequence ◽  
Alternative Means

Identification of Labeo rohita, L. bata and L. gonius is sometimes problematic when usual morphological features are lost and it is difficult to differentiate them with traditional morphological features at their diverse developmental stages. PCR-sequencing provides an authentic alternative means of identification of individuals at species level. Three local carp fishes were collected and 16S rRNA genes were sequenced by sanger sequencing method after PCR amplification using universal primers. Obtained sequences were found accurate with blast search result which showed maximum range of similarity with the existing respective gene fragments present in GenBank database. Sequences were compared and multiple sequence alignment has revealed some polymorphic sites which can be used to differentiate these three species from one another. This study may provide valuable understanding to study their population in future. Dhaka Univ. J. Biol. Sci. 26(2): 167-174, 2017 (July)

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