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Journal of Clinical Laboratory Analysis Mar 2014Identification of dermatophytes at the species level, relying on macro- and microscopic properties of the colonies is time-consuming, questioned in many circumstances,...
BACKGROUND
Identification of dermatophytes at the species level, relying on macro- and microscopic properties of the colonies is time-consuming, questioned in many circumstances, and requires considerable expertise. In this study, we examined the potency of a new genetic marker, β-tubulin (BT2) gene, for differentiation of dermatophytes in an in silico and experimental restriction fragment length polymorphism (RFLP) profile.
METHODS
The BT2 sequences of dermatophyte species were retrieved from GenBank and analyzed using bioinformatics softwares to choose suitable restriction enzyme(s). Forty reference culture collections and 100 clinical isolates were PCR-amplified using the primers T1 and Bt2b and consequently subjected to virtual RFLP analysis. The dermatophytes were identified according to specific lengths of bands in agarose gel electrophoresis.
RESULTS
After digestion of partially amplified β-tubulin gene with the restriction enzyme FatI, three dermatophyte species, that is, Microsporum gypseum, M. canis, and Trichophyton verrucosum yielded unique restriction maps while the remaining species including T. interdigitale, T. rubrum, T. tonsurans, T. schoenleinii, and T. violaceum, were identified by further restriction digestion by Alw21I, MwoI, and HpyCH4V endonucleases. The length of RFLP products was same as of those expected by computer analysis.
CONCLUSION
The two-step BT2 restriction mapping used in this study is an effective tool for reliable differentiation of the clinically relevant species of dermatophytes.
Topics: Arthrodermataceae; Humans; Polymorphism, Restriction Fragment Length; Restriction Mapping; Tubulin
PubMed: 24395510
DOI: 10.1002/jcla.21649 -
BMC Genomics Aug 2016Restriction site associated DNA sequencing (RAD-seq), a next-generation sequencing technology, has greatly facilitated genetic linkage mapping studies in outbred...
BACKGROUND
Restriction site associated DNA sequencing (RAD-seq), a next-generation sequencing technology, has greatly facilitated genetic linkage mapping studies in outbred species. RAD-seq is capable of discovering thousands of genetic markers for linkage mapping across many individuals, and can be applied in species with or without a reference genome. Although several analytical tools are available for RAD-seq data, alternative strategies are necessary for improving the marker quality and hence the genetic mapping accuracy.
RESULTS
We demonstrate a strategy for constructing dense genetic linkage maps in hybrid forest trees by combining RAD-seq and whole-genome sequencing technologies. We performed RAD-seq of 150 progeny and whole-genome sequencing of the two parents in an F1 hybrid population of Populus deltoides × P. simonii. Two rough references were assembled from the whole-genome sequencing reads of the two parents separately. Based on the parental reference sequences, 3442 high-quality single nucleotide polymorphisms (SNPs) were identified that segregate in the ratio of 1:1. The maternal linkage map of P. deltoides was constructed with 2012 SNPs, containing 19 linkage groups and spanning 4067.16 cM of the genome with an average distance of 2.04 cM between adjacent markers, while the male map of P. simonii consisted of 1430 SNPs and the same number of linkage groups with a total length of 4356.04 cM and an average interval distance of 3.09 cM. Collinearity between the parental linkage maps and the reference genome of P. trichocarpa was also investigated. Compared with the result on the basis of the existing reference genome, our strategy identified more high-quality SNPs and generated parental linkage groups that nicely match the karyotype of Populus.
CONCLUSIONS
The strategy of simultaneously using RAD and whole-genome sequencing technologies can be applied to constructing high-density genetic maps in forest trees regardless of whether a reference genome exists. The two parental linkage maps constructed here provide more accurate genetic resources for unraveling quantitative trait loci and accelerating molecular breeding programs, as well as for comparative genomics in Populus.
Topics: Chimera; Chromosome Mapping; Chromosomes, Plant; Genetic Linkage; Genome, Plant; Polymorphism, Single Nucleotide; Populus; Quantitative Trait Loci; Restriction Mapping; Sequence Analysis, DNA
PubMed: 27538483
DOI: 10.1186/s12864-016-3003-9 -
Intervirology 1989Physical maps were established for seven genome types of the intermediate adenovirus (AV) 15/H9 and for the serologically related prototypes AV15 and 9 using the enzymes... (Comparative Study)
Comparative Study
Physical maps were established for seven genome types of the intermediate adenovirus (AV) 15/H9 and for the serologically related prototypes AV15 and 9 using the enzymes BamHI, BglII, and HindIII. The polarity of the fragment order was determined by hybridization with known AV2 fragments. AV15/H9 strains showed more restriction sites in common with AV9 than with AV15. The results are consistent with the hypothesis that intermediate AV15/H9 strains have emerged by recombination.
Topics: Adenoviruses, Human; DNA, Viral; Genes, Viral; Nucleic Acid Hybridization; Recombination, Genetic; Restriction Mapping
PubMed: 2753655
DOI: 10.1159/000150089 -
Methods in Molecular Biology (Clifton,... 2011Restriction landmark genome scanning (RLGS) method is a high-resolution two-dimensional electrophoresis system for analyses of the whole genome DNA which is including...
Restriction landmark genome scanning (RLGS) method is a high-resolution two-dimensional electrophoresis system for analyses of the whole genome DNA which is including methylation status. It has been used for cloning genes of model animals and human genomes, detection of imprinted genes, and genome-wide methylation research in cancer. The conventional RLGS detected both polymorphism and methylated NotI sites between samples. Here, we have developed improved RLGS method with isoschizomer restriction enzymes such as MspI and HpaII to specifically detect methylated sites, using differential sensitivity of the restriction enzymes to methylated sequences. Recently, by using the genome database information, the RLGS spot sites were efficiently identified by this improved method. Then, genome methylation sites of Arabidopsis were mapped, and a unique inheritance was detected in methylated gene in rice. Now, epigenetic research becomes easy with the improved RLGS and it also can be applied for animal genome. Therefore, RLGS method is useful to explore for novel epigenetic phenomenon.
Topics: Arabidopsis; Autoradiography; DNA Methylation; DNA, Plant; Electrophoresis, Gel, Two-Dimensional; Genomics; Oryza; Restriction Mapping
PubMed: 21913074
DOI: 10.1007/978-1-61779-316-5_8 -
Journal of Computational Biology : a... 1998A fundamentally new molecular-biology approach in constructing restriction maps, Optical Mapping, has been developed by Schwartz et al. (1993). Using this method...
A fundamentally new molecular-biology approach in constructing restriction maps, Optical Mapping, has been developed by Schwartz et al. (1993). Using this method restriction maps are constructed by measuring the relevant fluorescence intensity and length measurements. However, it is difficult to directly estimate the restriction site locations of single DNA molecules based on these optical mapping data because of the precision of length measurements and the unknown number of true restriction sites in the data. We propose the use of a hierarchical Bayes model based on a mixture model with normals and random noise. In this model we explicitly consider the missing observation structure of the data, such as the orientations of molecules, the allocations of cutting sites to restriction sites, and the indicator variables of whether observed cut sites are true or false. Because of the complexity of the model, the large number of missing data, and the unknown number of restriction sites, we use Reversible-Jump Markov Chain Monte Carlo (MCMC) to estimate the number and the locations of the restriction sites. Since there exists a high multimodality due to unknown orientations of molecules, we also use a combination of our MCMC approach and the flipping algorithm suggested by Dancík and Waterman (1997). The study is highly computer-intensive and the development of an efficient algorithm is required.
Topics: Algorithms; Markov Chains; Models, Genetic; Monte Carlo Method; Restriction Mapping
PubMed: 9773346
DOI: 10.1089/cmb.1998.5.505 -
Methods in Molecular Biology (Clifton,... 2021NANOG is an embryonic transcription factor, which gets reexpressed in cancer stem or tumor initiating cells. NANOGP8, a retrogene belonging to the NANOG family, is... (Comparative Study)
Comparative Study
NANOG is an embryonic transcription factor, which gets reexpressed in cancer stem or tumor initiating cells. NANOGP8, a retrogene belonging to the NANOG family, is predominantly expressed in cancer cells and shows very high similarity with NANOG both at the nucleotide and at the protein level. The high similarity makes it extremely challenging to distinguish between these two transcription factors. Here we describe a highly efficient restriction endonuclease-based assay, which is performed on cDNA and allows to distinguish NANOGP8 from NANOG. This assay is critical to understand the specific role of NANOGP8 in cancer stemness, which in turn helps to unravel the therapeutic potential of targeting this undruggable transcription factor through gene therapy, for treatment of various cancers.
Topics: Biomarkers, Tumor; Cell Line, Tumor; DNA, Complementary; Deoxyribonucleases, Type II Site-Specific; Electrophoresis, Agar Gel; Humans; Nanog Homeobox Protein; Neoplasms; Restriction Mapping; Reverse Transcriptase Polymerase Chain Reaction; Sequence Alignment; Sequence Homology, Nucleic Acid; Substrate Specificity
PubMed: 34165720
DOI: 10.1007/978-1-0716-1503-4_16 -
Turkish Journal of Medical Sciences Oct 2018Background/aim: The identification of Candida species isolated from clinical specimens provides information about antifungal susceptibility and sheds light on the choice...
Background/aim: The identification of Candida species isolated from clinical specimens provides information about antifungal susceptibility and sheds light on the choice of empirical treatment. In the present study, restriction enzyme analysis of C. albicans and non-albicans Candida species previously identified by conventional methods was done to evaluate the utility of restriction enzyme analysis for more rapid and reliable identification of Candida species. Materials and methods: A total of 146 Candida strains isolated from various clinical specimens and ATCC strains were included. PCR products were digested with MwoI for all species and with BslI for C. parapsilosis and C. tropicalis strains. Results: The strains were identified by conventional methods as 40 C. albicans, 27 C. parapsilosis, 26 C. tropicalis, 25 C. glabrata, 11 C. kefyr, 10 C. krusei, and 7 C. guilliermondii strains. Restriction digestion with MwoI was able to distinguish between five different species (C. albicans, C. krusei, C. guilliermondii, C. kefyr, and C. glabrata), while BslI digestion could distinguish between C. tropicalis and C. parapsilosis. Conclusion: Restriction enzyme analysis with MwoI and BslI can be used for the identification of Candida species in situations where rapid identification is necessary or conventional methods are problematic.
Topics: Candida; Candidiasis; Humans; Polymerase Chain Reaction; Restriction Mapping
PubMed: 30384576
DOI: 10.3906/sag-1802-11 -
Research in Microbiology May 1997In the present study, 60 avian Chlamydia psittaci isolates were characterized using restriction fragment length polymorphism as well as serovar-specific monoclonal... (Comparative Study)
Comparative Study
In the present study, 60 avian Chlamydia psittaci isolates were characterized using restriction fragment length polymorphism as well as serovar-specific monoclonal antibodies, enabling a comparison between the two characterization methods. Sixty avian C. psittaci isolates were characterized by Alul restriction mapping of the major outer membrane protein gene omp1 obtained after amplification by the polymerase chain reaction. The 60 avian C. psittaci strains were also characterized using serovar-specific monoclonal antibodies in a microimmunofluorescence test. Digestion of 60 avian C. psittaci omp1 amplicons by Alul generated 5 of the 6 known distinct restriction patterns (A, B, D, E and F). Restriction pattern C was not observed. Serotyping revealed 4 avian C. psittaci serovars (A, B, C and D). None of the 60 isolates was typed as serovar E. AluI restriction patterns A, B, D and E corresponded in 98% of the cases to serovars A, B, C and D, respectively. One isolate, classified as serovar A, generated restriction pattern F instead of A. Genotyping enabled a more precise differentiation of avian C. psittaci serovar A strains. Serovar A strains were divided into two groups according to their Alul restriction pattern (A or F). For epidemiological studies, genotyping can thus be a highly valuable alternative to serotyping, especially when applied directly to the clinical samples.
Topics: Animals; Antibodies, Bacterial; Antibodies, Monoclonal; Bacterial Outer Membrane Proteins; Birds; Chlamydophila psittaci; Deoxyribonucleases, Type II Site-Specific; Fluorescent Antibody Technique; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Restriction Mapping; Serotyping
PubMed: 9765811
DOI: 10.1016/S0923-2508(97)81588-4 -
Journal of Clinical Microbiology Sep 1995
Review
Topics: Bacteria; Bacterial Typing Techniques; DNA, Bacterial; Electrophoresis, Gel, Pulsed-Field; Restriction Mapping
PubMed: 7494007
DOI: 10.1128/jcm.33.9.2233-2239.1995 -
Nature Reviews. Genetics Feb 2016High-throughput techniques based on restriction site-associated DNA sequencing (RADseq) are enabling the low-cost discovery and genotyping of thousands of genetic... (Review)
Review
High-throughput techniques based on restriction site-associated DNA sequencing (RADseq) are enabling the low-cost discovery and genotyping of thousands of genetic markers for any species, including non-model organisms, which is revolutionizing ecological, evolutionary and conservation genetics. Technical differences among these methods lead to important considerations for all steps of genomics studies, from the specific scientific questions that can be addressed, and the costs of library preparation and sequencing, to the types of bias and error inherent in the resulting data. In this Review, we provide a comprehensive discussion of RADseq methods to aid researchers in choosing among the many different approaches and avoiding erroneous scientific conclusions from RADseq data, a problem that has plagued other genetic marker types in the past.
Topics: Biological Evolution; Genomics; High-Throughput Nucleotide Sequencing; Humans; Metagenomics; Restriction Mapping
PubMed: 26729255
DOI: 10.1038/nrg.2015.28