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BioTechniques Sep 2002Ever since methylcytosine was found in genomic DNA, this epigenetic alteration has become a center of scientific attraction, especially because of its relation to gene... (Review)
Review
Ever since methylcytosine was found in genomic DNA, this epigenetic alteration has become a center of scientific attraction, especially because of its relation to gene silencing in disease. There is currently a wide range of methods designed to yield quantitative and qualitative information on genomic DNA methylation. The earliest approaches were concentrated on the study of overall levels of methylcytosine, but more recent efforts havefocused on the study ofthe methylation status of specific DNA sequences. Particularly, optimization of the methods based on bisulfite modification of DNA permits the analysis of limited CpGs in restriction enzyme sites (e.g., combined bisulfite restriction analyses and methylation-sensitive single nucleotide primer extension) and the overall characterization based on differential methylation states (e.g., methylation-specific PCR, MethyLight, and methylation-sensitive single-stranded conformational polymorphism) and allows very specific patterns of methylation to be revealed (bisulfite DNA sequencing). In addition, novel methods designed to search for new methylcytosine hot spots have yielded further data without requiring prior knowledge of the DNA sequence. We hope this review will be a valuable tool in selecting the best techniques to address particular questions concerning the cytosine methylation status of genomic DNA.
Topics: 5-Methylcytosine; Chromatography, High Pressure Liquid; Cytosine; DNA; DNA Methylation; Electrophoresis, Capillary; Genome; Microchemistry; Polymerase Chain Reaction; Restriction Mapping; Sequence Analysis, DNA; Sulfites
PubMed: 12238773
DOI: 10.2144/02333rv01 -
Current Issues in Molecular Biology 1999The polymerase chain reaction (PCR) is the most widely used technique for the study of DNA. Applications for PCR have been extended significantly by the development of...
The polymerase chain reaction (PCR) is the most widely used technique for the study of DNA. Applications for PCR have been extended significantly by the development of "long" PCR, a technique that makes it possible to amplify DNA fragments up to 40 kb in length. This article describes two novel applications of the long PCR technique, one which simplifies restriction mapping and another which enhances amplification specificity and yield. The same primers used to perform the long PCR amplification can be used as probes to perform restriction mapping of the DNA fragment amplified. Restriction digestion performed prior to long PCR amplification can be used to selectively suppress the amplification of members of families of closely related DNA sequences, thereby making it possible to selectively amplify one of a group of highly homologous sequences. These two complimentary techniques, both involving use of the long PCR paired with restriction digestion, have potential application in any laboratory in which PCR is performed.
Topics: DNA Restriction Enzymes; Humans; Polymerase Chain Reaction; Restriction Mapping
PubMed: 11475703
DOI: No ID Found -
Applied and Environmental Microbiology Apr 1990Plasmid DNAs from 15 Propionibacterium strains were characterized by using restriction endonuclease analyses, DNA-DNA hybridizations, and curing experiments. Restriction...
Plasmid DNAs from 15 Propionibacterium strains were characterized by using restriction endonuclease analyses, DNA-DNA hybridizations, and curing experiments. Restriction endonuclease analysis identified seven distinct plasmids (pRGO1 through pRGO7). Detailed restriction maps were constructed for four of these plasmids. DNA-DNA hybridization analysis revealed that plasmids pRGO1 and pRGO2 had extensive sequence homology and that both were homologous to pRGO7 and to similar sequences of pRGO5. Plasmids pRGO4 and pRGO6 did not have any significant sequence homology with any of the other plasmids. Plasmid pRGO3 had partial sequence homology only with pRGO7. Curing of plasmids pRGO1, pRGO2, and pRGO5 was achieved by treatment with acriflavin, but we failed to identify any plasmid-encoded bacteriocin production, carbohydrate fermentation, or antibiotic resistance. However, physical evidence was obtained that tentatively linked the clumping phenotype of Propionibacterium jensenii P38 with plasmid pRGO5.
Topics: Base Sequence; DNA, Bacterial; Nucleic Acid Hybridization; Plasmids; Propionibacterium; Restriction Mapping
PubMed: 2339876
DOI: 10.1128/aem.56.4.864-871.1990 -
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 -
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 -
Bioinformatics (Oxford, England) Mar 2023Efficient tapping into genomic information from a single microscopic image of an intact DNA molecule is an outstanding challenge and its solution will open new frontiers...
MOTIVATION
Efficient tapping into genomic information from a single microscopic image of an intact DNA molecule is an outstanding challenge and its solution will open new frontiers in molecular diagnostics. Here, a new computational method for optical genome mapping utilizing deep learning is presented, termed DeepOM. Utilization of a convolutional neural network, trained on simulated images of labeled DNA molecules, improves the success rate in the alignment of DNA images to genomic references.
RESULTS
The method is evaluated on acquired images of human DNA molecules stretched in nano-channels. The accuracy of the method is benchmarked against state-of-the-art commercial software Bionano Solve. The results show a significant advantage in alignment success rate for molecules shorter than 50 kb. DeepOM improves the yield, sensitivity, and throughput of optical genome mapping experiments in applications of human genomics and microbiology.
AVAILABILITY AND IMPLEMENTATION
The source code for the presented method is publicly available at https://github.com/yevgenin/DeepOM.
Topics: Humans; Deep Learning; Genomics; Restriction Mapping; Software; DNA; Genome, Human
PubMed: 36929928
DOI: 10.1093/bioinformatics/btad137 -
Proceedings of the National Academy of... Jun 1992The diversity of chemical synthesis and the power of genetics are linked to provide a powerful, versatile method for drug screening. A process of alternating parallel...
The diversity of chemical synthesis and the power of genetics are linked to provide a powerful, versatile method for drug screening. A process of alternating parallel combinatorial synthesis is used to encode individual members of a large library of chemicals with unique nucleotide sequences. After the chemical entity is bound to a target, the genetic tag can be amplified by replication and utilized for enrichment of the bound molecules by serial hybridization to a subset of the library. The nature of the chemical structure bound to the receptor is decoded by sequencing the nucleotide tag.
Topics: Amino Acid Sequence; Base Sequence; Databases, Factual; Drug Design; Molecular Sequence Data; Peptides; Polymerase Chain Reaction; Restriction Mapping
PubMed: 1608946
DOI: 10.1073/pnas.89.12.5381 -
Genetics Nov 2011Forward genetic screens provide a powerful approach for inferring gene function on the basis of the phenotypes associated with mutated genes. However, determining the...
Forward genetic screens provide a powerful approach for inferring gene function on the basis of the phenotypes associated with mutated genes. However, determining the causal mutation by traditional mapping and candidate gene sequencing is often the rate-limiting step, especially when analyzing many mutants. We report two genomic approaches for more rapidly determining the identity of the affected genes in Caenorhabditis elegans mutants. First, we report our use of restriction site-associated DNA (RAD) polymorphism markers for rapidly mapping mutations after chemical mutagenesis and mutant isolation. Second, we describe our use of genomic interval pull-down sequencing (GIPS) to selectively capture and sequence megabase-sized portions of a mutant genome. Together, these two methods provide a rapid and cost-effective approach for positional cloning of C. elegans mutant loci, and are also applicable to other genetic model systems.
Topics: Animals; Caenorhabditis elegans; DNA; DNA Mutational Analysis; Genetic Loci; Genome; Polymorphism, Genetic; Restriction Mapping
PubMed: 21900274
DOI: 10.1534/genetics.111.134031 -
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 -
International Journal For Parasitology Jul 2009The development of forward genetics as a functional system in Toxoplasma gondii spanned more than three decades from the mid-1970s until now. The initial demonstration... (Review)
Review
The development of forward genetics as a functional system in Toxoplasma gondii spanned more than three decades from the mid-1970s until now. The initial demonstration of experimental genetics relied on chemically induced drug-resistant mutants that were crossed by co-infecting cats, collecting oocysts, sporulating and hatching progeny in vitro. To capitalise on this, genetic markers were employed to develop linkage maps by tracking inheritance through experimental crosses. In all, three generations of genetic maps were developed to define the chromosomes, estimate recombination rates and provide a system for linkage analysis. Ultimately this genetic map would become the foundation for the assembly of the T. gondii genome, which was derived from whole genome shotgun sequencing, into a chromosome-centric view. Finally, application of forward genetics to multigenic biological traits showed the potential to map and identify specific genes that control complex phenotypes including virulence.
Topics: Animals; Cats; Chromosome Mapping; Crosses, Genetic; Drug Resistance; Genetics, Population; Genome, Protozoan; History, 20th Century; Humans; Life Cycle Stages; Mice; Phylogeny; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Protozoan Proteins; Restriction Mapping; Toxoplasma; Toxoplasmosis; Virulence
PubMed: 19254720
DOI: 10.1016/j.ijpara.2009.02.011