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Methods in Molecular Biology (Clifton,... 2018Digital Restriction Enzyme Analysis of Methylation (DREAM) is a method for quantitative mapping of DNA methylation across genomes using next-generation sequencing (NGS)...
Digital Restriction Enzyme Analysis of Methylation (DREAM) is a method for quantitative mapping of DNA methylation across genomes using next-generation sequencing (NGS) technology. The method is based on sequential cuts of genomic DNA with a pair of restriction enzymes (SmaI and XmaI) at CCCGGG target sites. Unmethylated sites are first digested with SmaI. This enzyme cuts the sites in the middle at CCC^GGG, leaving behind blunt ended fragments. CpG methylation completely blocks SmaI; therefore, only unmethylated sites are cleaved. The remaining methylated sites are digested with XmaI in the next step. This enzyme is not blocked by CpG methylation. It cuts the recognition site sideways at C^CCGGG forming 5'-CCGG overhangs. The sequential cuts thus create distinct methylation-specific signatures at the ends of restriction fragments: 5'-GGG for unmethylated CpG sites and 5'-CCGGG for methylated sites. The DNA fragments resulting from the digestions are ligated to NGS adapters. Sequencing libraries are prepared using hexanucleotide barcodes for sample identification. Individual libraries with distinct barcodes are pooled and sequenced using a paired ends protocol. The sequencing reads are aligned to the genome and mapped to unique CCCGGG target sites. Methylation at individual CpG sites is calculated as the ratio of sequencing reads with the methylated signature to the total number of reads mapping to the site. Sequencing of 25 million reads per sample typically yields 50,000 unique CpG sites covered with hundreds of reads enabling accurate determination of DNA methylation levels. DREAM does not require bisulfite conversion, has a very low background, and has high sensitivity to low levels of methylation. The method is simple, cost-effective, quantitative, highly reproducible, and can be applied to any species.
Topics: CpG Islands; DNA Methylation; DNA Restriction Enzymes; Genome, Human; Genomic Library; High-Throughput Nucleotide Sequencing; Humans; Restriction Mapping; Sequence Analysis, DNA
PubMed: 29224148
DOI: 10.1007/978-1-4939-7481-8_13 -
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 -
Mycological Research Jan 2005Accurate identification of Alternaria spp. is dependent upon the production of diagnostic morphological characters under defined cultural conditions and the proper...
Accurate identification of Alternaria spp. is dependent upon the production of diagnostic morphological characters under defined cultural conditions and the proper assessment of character variation. This process is often compromised by variation in laboratory facilities and technical expertise. To assist taxon identification and phylogenetic studies, restriction site information from the intergenic spacer (IGS) region of nuclear rDNA was evaluated. Restriction maps were constructed from 15 species of Alternaria and Stemphylium botryosum (telemorph Pleospora herbarum) for 11 restriction enzymes using a new method for restriction mapping based on differential priming of IGS amplicons. IGS fragment size varied among species from 2.2-3.9 kb. Based upon restriction site homology among closely-related and more distantly related species, the IGS region could be divided into conserved and variable domains. The conserved domain was approximately 0.75 kb in size and was located at the 3' end of the IGS region. Restriction site homology within this region was very high, especially among closely related taxa. The remainder of the region comprised the variable domain, which encompassed considerable differences in size and restriction sites among taxa. The presence or absence of restriction sites among taxa was analyzed using methods of neighbor-joining. Phylogenetic relationships based on this method were concordant with those previously resolved based upon other methods and other genomic regions.
Topics: Alternaria; DNA, Fungal; DNA, Ribosomal Spacer; Mycological Typing Techniques; Phylogeny; Polymerase Chain Reaction; Restriction Mapping; Sensitivity and Specificity; Species Specificity
PubMed: 15736866
DOI: 10.1017/s0953756204001388 -
PloS One 2021In genomics, optical mapping technology provides long-range contiguity information to improve genome sequence assemblies and detect structural variation. Originally a...
In genomics, optical mapping technology provides long-range contiguity information to improve genome sequence assemblies and detect structural variation. Originally a laborious manual process, Bionano Genomics platforms now offer high-throughput, automated optical mapping based on chips packed with nanochannels through which unwound DNA is guided and the fluorescent DNA backbone and specific restriction sites are recorded. Although the raw image data obtained is of high quality, the processing and assembly software accompanying the platforms is closed source and does not seem to make full use of data, labeling approximately half of the measured signals as unusable. Here we introduce two new software tools, independent of Bionano Genomics software, to extract and process molecules from raw images (OptiScan) and to perform molecule-to-molecule and molecule-to-reference alignments using a novel signal-based approach (OptiMap). We demonstrate that the molecules detected by OptiScan can yield better assemblies, and that the approach taken by OptiMap results in higher use of molecules from the raw data. These tools lay the foundation for a suite of open-source methods to process and analyze high-throughput optical mapping data. The Python implementations of the OptiTools are publicly available through http://www.bif.wur.nl/.
Topics: Chromosome Mapping; Genomics; High-Throughput Nucleotide Sequencing; Optical Restriction Mapping; Sequence Analysis, DNA
PubMed: 34591846
DOI: 10.1371/journal.pone.0253102 -
Tanpakushitsu Kakusan Koso. Protein,... Feb 1993
Review
Topics: Chromosomes, Human, Pair 21; DNA; Deoxyribonucleases, Type II Site-Specific; Genetic Linkage; Humans; Restriction Mapping
PubMed: 8488261
DOI: No ID Found -
Computer Applications in the... Jun 1994Eight important restriction mapping programs, developed between 1978 and 1993, are analyzed and their performance evaluated. The analyses concentrate on the practical...
Eight important restriction mapping programs, developed between 1978 and 1993, are analyzed and their performance evaluated. The analyses concentrate on the practical value of the programs to molecular biologists who do restriction mapping on a daily basis, rather than on theoretical efficiency. Although all of the programs could find maps consistent with the data, none were able to discriminate reliably the true map from the other consistent maps, given realistic levels of error. To alleviate this problem, we propose an expert mapping system which utilizes the same inferences and mapping techniques used by expert human mappers to reduce the number of false maps found.
Topics: Algorithms; Evaluation Studies as Topic; Forecasting; History, 20th Century; Molecular Biology; Reproducibility of Results; Restriction Mapping; Software
PubMed: 7922680
DOI: 10.1093/bioinformatics/10.3.249 -
Bioinformatics (Oxford, England) 1998To determine the most powerful artificial intelligence techniques for automated restriction mapping, and use them to create a powerful multiple-enzyme restriction... (Comparative Study)
Comparative Study
MOTIVATION
To determine the most powerful artificial intelligence techniques for automated restriction mapping, and use them to create a powerful multiple-enzyme restriction mapping tool.
RESULTS
The most effective search engine utilized model-driven exhaustive search and a form of binary logic pruning based on Pratt's separation theory. Additional experimentation led to the development of an input preprocessing module which significantly speeds up searches, and an output post-processing module which enables users to analyze large solution sets and reduce their apparent complexity.
AVAILABILITY
An executable version of the resultant tool, Mapper, can be downloaded from our Web site (http://www.ai.eecs.uic.edu) by selecting the 'Software' option.
CONTACT
[email protected] (http://www.ai.eecs.uic.edu/ñelson).
Topics: Algorithms; Artificial Intelligence; Computational Biology; Evaluation Studies as Topic; Restriction Mapping; Software
PubMed: 9545441
DOI: 10.1093/bioinformatics/14.2.101 -
PCR Methods and Applications Feb 1993Although direct DNA sequencing is now readily available, restriction enzyme analyses are still widely used in population genetics and molecular systematics studies....
Although direct DNA sequencing is now readily available, restriction enzyme analyses are still widely used in population genetics and molecular systematics studies. These analyses provide cheaper and faster ways to assay patterns of nucleotide differentiation across a large number of individuals. In this paper, we introduce a new approach to restriction enzyme analyses in which high-resolution restriction site maps are obtained from partial digestions of PCR products. This procedure increases the level of resolution at least an order of magnitude over the double-digestion method for restriction enzyme mapping, can target specific DNA regions with the use of specific primers, and, because it uses chemiluminescent detection of DNA, can be easily implemented in laboratories that lack the necessary setups to handle radioactive substances.
Topics: Animals; Biotin; DNA, Mitochondrial; Luminescent Measurements; Polymerase Chain Reaction; Restriction Mapping
PubMed: 8443575
DOI: 10.1101/gr.2.3.228 -
DNA Sequence : the Journal of DNA... 2000Mitochondrial DNA was isolated from samples of the four-wing flyingfish, Hirundichthys affinis, collected in Barbados in January 1996 and subjected to restriction enzyme...
Mitochondrial DNA was isolated from samples of the four-wing flyingfish, Hirundichthys affinis, collected in Barbados in January 1996 and subjected to restriction enzyme analysis, using 13 restriction endonucleases which recognise hexanucleotide sequences, in single and double digests. The resulting restricted DNA fragments were used to map the 14 enzyme recognition sites of 6 endonucleases (7 had no sites) on the flyingfish mtDNA molecule for the first time. In addition, the mtDNA D-loop region was positioned on the restriction site map, for the first time, by selective restriction digestion of the mtDNA molecule followed by polymerase chain reaction (PCR) amplification of the resulting fragments using specific mtDNA D-loop primers. The size of the flyingfish mtDNA molecule (18 kb) was also determined.
Topics: Animals; Barbados; DNA Restriction Enzymes; DNA, Mitochondrial; Fishes; Nucleic Acid Conformation; Polymerase Chain Reaction; Restriction Mapping
PubMed: 11092740
DOI: 10.3109/10425170009033243 -
Current Opinion in Biotechnology Feb 1997The specific protection of only one of many restriction sites in a genome from inactivation by a cognate methyltransferase (MTase) creates a unique cleavage site - an... (Review)
Review
The specific protection of only one of many restriction sites in a genome from inactivation by a cognate methyltransferase (MTase) creates a unique cleavage site - an Achilles' heel cleavage (AC) site. In the RecA-AC, or RARE, technique, such specific protection is provided by a synaptic complex composed of RecA protein, a gamma-S analog of ATP and a 30-60 nucleotide long oligodeoxynucleotide complementary or identical to the sequence-targeted site in which the protected restriction site is embedded. Upon methylation and the subsequent removal of the protective complex and MTase, the protected site is the only site cut by the cognate restriction enzyme. Two such targeted cuts permit the excision of a unique DNA fragment from the genome. Recent advances include the calibration of DNA clones, the mapping of gaps, and the determination of the sizes of excised fragments by pulsed-field gel electrophoresis, which allows one to measure distances between any two neighboring sequence-targeted sites, in the range of a few kilobases to 10 megabases, with the purpose of physically mapping the genome.
Topics: Binding Sites; Biotechnology; DNA; DNA Restriction Enzymes; Rec A Recombinases; Restriction Mapping
PubMed: 9013652
DOI: 10.1016/s0958-1669(97)80161-9