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Experimental Gerontology May 2005Telomeres provide an essential 'capping' function, which prevents the natural end of a chromosome from being recognised as a simple dsDNA break. The biology of human... (Review)
Review
Telomeres provide an essential 'capping' function, which prevents the natural end of a chromosome from being recognised as a simple dsDNA break. The biology of human telomeres is inextricably linked with both cancer and ageing. As such there is considerable interest in determining the length of these essential and dynamic structures. Here I review, from the standpoint of ageing research in humans, the current situation with regards to technologies available to determine telomere length in human cells and tissues.
Topics: Base Sequence; DNA; Humans; In Situ Hybridization, Fluorescence; Polymerase Chain Reaction; Restriction Mapping; Telomere
PubMed: 15919587
DOI: 10.1016/j.exger.2005.02.008 -
Analytical Biochemistry Aug 2002The field of molecular biology was revolutionized by the advent of gel electrophoresis. Restriction landmark genomic scanning (RLGS) is a type of two-dimensional... (Review)
Review
The field of molecular biology was revolutionized by the advent of gel electrophoresis. Restriction landmark genomic scanning (RLGS) is a type of two-dimensional electrophoresis employed in the genome-wide assessment of genomic alterations. RLGS has been used to study genetic and epigenetic changes in normal tissues, primary tumors, cancer cell lines, and various organisms such as mice, rats, hamsters, bacteria, and plants. An RLGS profile displays over 2000 radiolabeled restriction landmark sites in a single assay. When conducted with methylation-sensitive restriction enzymes whose sites are preferentially located in CpG island regulatory regions, RLGS becomes a very versatile tool for the investigation of both normal and aberrant methylation patterns. Early studies performed on tumor DNA were mainly descriptive in nature, essentially a catalogue of loci that were changed to varying degrees in different tumor types. Over time, as investigators have become more proficient with RLGS and have undertaken high-throughput studies, the need for efficient cloning, imaging, and analysis systems has become paramount. Current studies focus on identifying specific genes and pathways involved in deregulated methylation in cancer. As such, RLGS analysis of tumor samples has made tremendous contributions to our understanding of the role of DNA methylation in cancer. Future directions will take advantage of the abundant genomic sequence data available to link all of the RLGS loci to genes and create biologically relevant methylation profiles of cancer. This review discusses practical considerations of using RLGS as a genome scanning tool and the past, present, and future applications in cancer biology.
Topics: Animals; Computational Biology; DNA Methylation; Electrophoresis, Gel, Two-Dimensional; Gene Expression Regulation, Neoplastic; Genomics; Neoplasms; Restriction Mapping
PubMed: 12202234
DOI: 10.1016/s0003-2697(02)00033-7 -
Analytical and Bioanalytical Chemistry Aug 2008Biological assays at the single molecule level are crucial to fundamental studies of DNA-protein mechanisms. In order to cater for high throughput applications, one area... (Review)
Review
Biological assays at the single molecule level are crucial to fundamental studies of DNA-protein mechanisms. In order to cater for high throughput applications, one area of immense research potential is single-molecule bioassays where miniaturized devices are developed to perform rapid and effective biological reactions and analyses. With the success of various emerging technologies for engineering miniaturized structures down to the nanoscale level, supported by specialized equipment for detection, many investigations in the field of life science that were once thought impossible can now be actively explored. In this review, the significance of downscaling to the single-molecule level is firstly presented in selected examples, with the focus placed on restriction enzyme assays. To determine the effectiveness of single-molecule restriction enzyme reactions, simple and direct analytical methods based on DNA stretching have often been reliably employed. DNA stretching can be realized based on a number of working principles related to the physical forces exerted on the DNA samples. We then discuss two examples of a nanochannel system and a microchamber system where single-molecule restriction enzyme digestion and DNA stretching have been integrated, which possess prospective capabilities of developing into highly sensitive and high-throughput restriction enzyme assays. Finally, we take a brief look at the general trends in technological development in this field by comparing the advantages and disadvantages of performing assays at bulk, microscale and single-molecule levels.
Topics: DNA; Microchemistry; Nanostructures; Nanotechnology; Nucleic Acid Conformation; Restriction Mapping; Sensitivity and Specificity
PubMed: 18427787
DOI: 10.1007/s00216-008-2099-4 -
Methods in Enzymology 1998
Topics: Animals; Bacteriophage M13; Cloning, Molecular; DNA Primers; Escherichia coli; Indicators and Reagents; Mutagenesis, Site-Directed; Plasmids; Polymerase Chain Reaction; Potassium Channels; Recombinant Fusion Proteins; Recombinant Proteins; Restriction Mapping
PubMed: 9711602
DOI: 10.1016/s0076-6879(98)93007-5 -
Bioinformatics (Oxford, England) Oct 2023Optical genome mapping (OGM) is a technique that extracts partial genomic information from optically imaged and linearized DNA fragments containing fluorescently labeled...
MOTIVATION
Optical genome mapping (OGM) is a technique that extracts partial genomic information from optically imaged and linearized DNA fragments containing fluorescently labeled short sequence patterns. This information can be used for various genomic analyses and applications, such as the detection of structural variations and copy-number variations, epigenomic profiling, and microbial species identification. Currently, the choice of labeled patterns is based on the available biochemical methods and is not necessarily optimized for the application.
RESULTS
In this work, we develop a model of OGM based on information theory, which enables the design of optimal labeling patterns for specific applications and target organism genomes. We validated the model through experimental OGM on human DNA and simulations on bacterial DNA. Our model predicts up to 10-fold improved accuracy by optimal choice of labeling patterns, which may guide future development of OGM biochemical labeling methods and significantly improve its accuracy and yield for applications such as epigenomic profiling and cultivation-free pathogen identification in clinical samples.
AVAILABILITY AND IMPLEMENTATION
https://github.com/yevgenin/PatternCode.
Topics: Humans; Information Theory; Software; Genome; Restriction Mapping; DNA
PubMed: 37758248
DOI: 10.1093/bioinformatics/btad601 -
Genome Research Dec 2002The development of statistical methodologies for quantitative trait locus (QTL) mapping in polyploids is complicated by complex polysomic inheritance. In this article,... (Comparative Study)
Comparative Study
The development of statistical methodologies for quantitative trait locus (QTL) mapping in polyploids is complicated by complex polysomic inheritance. In this article, we propose a statistical method for mapping QTL in tetraploids undergoing bivalent formation at meiosis by using single-dose restriction fragments. Our method is based on a unified framework, one that uses chromosome bivalent pairing configuration and gametic recombination to discern different mechanisms of gamete formation. Our bivalent polyploid model can not only provide a simultaneous estimation of the linkage and chromosome pairing configuration-a cytological parameter of evolutionary and systematic interest-but also enhances the precision of estimating QTL effects and position by correctly characterizing gene segregation during polyploid meiosis. By using our method and a linkage map constructed in a previous study, we successfully identify several QTL affecting winter hardiness in bivalent tetraploid alfalfa. Moreover, our results reveal significant preferential chromosome pairing at meiosis in an F1 hybrid population, which indicates the importance of reassessing the traditional view of random chromosome segregation in alfalfa.
Topics: Chromosome Pairing; Chromosome Segregation; Computational Biology; Computer Simulation; Genetic Markers; Medicago sativa; Meiosis; Models, Genetic; Models, Statistical; Polymorphism, Restriction Fragment Length; Polyploidy; Quantitative Trait Loci; Restriction Mapping
PubMed: 12466302
DOI: 10.1101/gr.320202 -
Genetics Nov 1994A set of 147 Dictyostelium discoideum strains was constructed by random integration of a vector containing rare restriction sites. The strains were generated by...
A set of 147 Dictyostelium discoideum strains was constructed by random integration of a vector containing rare restriction sites. The strains were generated by transformation using restriction enzyme-mediated integration (REMI) which results in the integration of linear DNA fragments into randomly distributed genomic restriction sites. Restriction fragment length polymorphism (RFLP) was generated in a single genomic site in each strain. These REMI-RFLP strains were used to confirm gene linkages previously supported by two other physical mapping techniques: yeast artificial chromosome (YAC) contig construction, and megabase-scale restriction mapping. New linkages were uncovered when two or more hybridization probes identified the same RFLP fragments. Probes for 100 genes have marked 53% of the RFLPs, representing greater than 22 Mb of the 40 Mb Dictyostelium genome. Alignment of these and other large fragments along each chromosome should lead to a complete physical map of the Dictyostelium genome.
Topics: Animals; Chromosomes, Artificial, Yeast; DNA Restriction Enzymes; Dictyostelium; Genome, Fungal; Multigene Family; Polymorphism, Restriction Fragment Length; Restriction Mapping
PubMed: 7851764
DOI: 10.1093/genetics/138.3.665 -
Methods in Molecular Biology (Clifton,... 2002
Topics: Deoxyribonucleases, Type II Site-Specific; Female; Hemophilia A; Humans; Indicators and Reagents; Male; Nucleic Acid Hybridization; Pedigree; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Restriction Mapping
PubMed: 12013748
DOI: 10.1385/1-59259-273-2:029 -
Methods in Molecular Medicine 2005Many tumor suppressor genes (such as p16, Rb, VHL, E-cadherin, and hMLH1) that are silenced by mutation are also inactivated by gene silencing through DNA methylation.... (Review)
Review
Many tumor suppressor genes (such as p16, Rb, VHL, E-cadherin, and hMLH1) that are silenced by mutation are also inactivated by gene silencing through DNA methylation. Characterization of genes hypermethylated in human cancers but not in normal tissues not only provides insights into cancer biology but also permits the use of methylation-specific polymerase chain reaction-based assays that could serve as diagnostic tests for the early detection and early diagnosis of this disease. To this end, research aimed at the identification and characterization of the methylation status of known and candidate tumor suppressor genes is one strategy for finding putative diagnostic markers. This chapter describes several methods of methylation analysis.
Topics: DNA Methylation; Genes, Tumor Suppressor; Humans; Neoplasms; Polymerase Chain Reaction; Restriction Mapping
PubMed: 15542902
DOI: 10.1385/1-59259-780-7:123 -
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