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Journal of Muscle Research and Cell... 2002Restriction enzyme-mediated integration (REMI) has been used to study a number of cellular and developmental processes in Dictyostelium discoideum. In this paper we... (Review)
Review
Restriction enzyme-mediated integration (REMI) has been used to study a number of cellular and developmental processes in Dictyostelium discoideum. In this paper we review the basics of this powerful method of introducing random mutations in Dictyostelium. Here we discuss several mutation screens that have been devised and some of the genes that have been discovered through this approach to mutagenesis. Included in this discussion is how one goes about isolating a gene that has been disrupted by REMI, and how one confirms that this disruption is actually responsible for the observed phenotype. Finally, we describe how REMI can be used as an effective teaching tool in undergraduate cell biology laboratory courses.
Topics: Animals; DNA Restriction Enzymes; Dictyostelium; Genes, Fungal; Genes, Protozoan; Models, Genetic; Mutagenesis; Polymerase Chain Reaction; Protozoan Proteins; Restriction Mapping
PubMed: 12952058
DOI: 10.1023/a:1024494704863 -
Applied and Environmental Microbiology Sep 2005Rhodospirillum rubrum is a phototrophic purple nonsulfur bacterium known for its unique and well-studied nitrogen fixation and carbon monoxide oxidation systems and as a...
Rhodospirillum rubrum is a phototrophic purple nonsulfur bacterium known for its unique and well-studied nitrogen fixation and carbon monoxide oxidation systems and as a source of hydrogen and biodegradable plastic production. To better understand this organism and to facilitate assembly of its sequence, three whole-genome restriction endonuclease maps (XbaI, NheI, and HindIII) of R. rubrum strain ATCC 11170 were created by optical mapping. Optical mapping is a system for creating whole-genome ordered restriction endonuclease maps from randomly sheared genomic DNA molecules extracted from cells. During the sequence finishing process, all three optical maps confirmed a putative error in sequence assembly, while the HindIII map acted as a scaffold for high-resolution alignment with sequence contigs spanning the whole genome. In addition to highlighting optical mapping's role in the assembly and confirmation of genome sequence, this work underscores the unique niche in resolution occupied by the optical mapping system. With a resolution ranging from 6.5 kb (previously published) to 45 kb (reported here), optical mapping advances a "molecular cytogenetics" approach to solving problems in genomic analysis.
Topics: Contig Mapping; Genome, Bacterial; Image Processing, Computer-Assisted; Restriction Mapping; Rhodospirillum rubrum; Sequence Analysis, DNA
PubMed: 16151144
DOI: 10.1128/AEM.71.9.5511-5522.2005 -
Methods in Molecular Biology (Clifton,... 1997
Topics: Restriction Mapping; Software
PubMed: 9089615
DOI: No ID Found -
Journal of Molecular Evolution Sep 1997Multiple band patterns of DNA repeats in the 20-500-nucleotide range can be detected by digesting genomic DNA with short-cutting restriction endonucleases, followed by...
Multiple band patterns of DNA repeats in the 20-500-nucleotide range can be detected by digesting genomic DNA with short-cutting restriction endonucleases, followed by end labeling of the restriction fragments and fractionation in nondenaturing polyacrylamide gels. We call such band patterns obtained from genomic DNA "taxonprints" (Fedorov et al. 1992). Here we show that taxonprints for the taxonomic groups studied (mammals, reptiles, fish, insects-altogether more than 50 species) have the following properties: (1) All individuals from the same species have identical taxonprints. (2) Taxonprint bands can be subdivided into those specific for a single species and those specific for groups of closely related species, genera, and even families. (3) Each restriction endonuclease produces unique band patterns; thus, five to ten restriction enzymes (about 100 bands) may be sufficient for a statistical treatment of phylogenetic relationships based on polymorphisms of restriction endinuclease sites. We demonstrate that taxonprint analysis allows one to distinguish closely related species and to establish the degree of similarity among species and among genera. These characteristics make taxonprint analysis a valuable tool for taxonomic and phylogenetic studies.
Topics: Animals; Classification; Deoxyribonucleases, Type II Site-Specific; Endonucleases; Genetic Markers; Hedgehogs; Humans; Phylogeny; Racial Groups; Repetitive Sequences, Nucleic Acid; Restriction Mapping; Species Specificity
PubMed: 9302328
DOI: 10.1007/pl00006237 -
Preventive Veterinary Medicine Sep 1997A quantitative algorithm for comparing restriction fragment patterns (RFPs) was developed and used to estimate the genomic similarity of 18 isolates of pseudorabies... (Comparative Study)
Comparative Study Review
A quantitative algorithm for comparing restriction fragment patterns (RFPs) was developed and used to estimate the genomic similarity of 18 isolates of pseudorabies virus of known origin. Variants of this algorithm using either untransformed or square-root-transformed differences between fragment sizes were investigated with regard to their ability to classify RFPs. Multidimensional scaling was used to represent spatially the genomic relatedness among samples, with 3 dimensions producing the most meaningful results. The square-root transformation provided more interpretable dimensions. The first dimension differentiated samples geographically, separating North American from European isolates. The second and third dimensions differentiated isolates with specific gene deletions (gE and gG, respectively) from those not having these deletions. Clusters of isolates were identified that were related either by collection from the same geographic area during a specific time period, or by laboratory intervention to create vaccines. These methods offer increased precision in the determination of genetic relatedness based on RFPs, and thus offer increased diagnostic accuracy for the determination of sources of infection.
Topics: Algorithms; Animals; DNA, Viral; Genome, Viral; Herpesvirus 1, Suid; Molecular Epidemiology; Restriction Mapping
PubMed: 9361323
DOI: 10.1016/s0167-5877(96)01134-8 -
Proceedings. International Conference... 1999In this paper, we describe our algorithmic approach to constructing an alignment of (contiging) a set of restriction maps created from the images of individual genomic...
In this paper, we describe our algorithmic approach to constructing an alignment of (contiging) a set of restriction maps created from the images of individual genomic (uncloned) DNA molecules digested by restriction enzymes. Generally, these DNA segments are sized in the range of 1-4 Mb. The goal is to devise contiging algorithms capable of producing high-quality composite maps rapidly and in a scaleable manner. The resulting software is a key component of our physical mapping automation tools and has been used to create complete maps of various microorganisms (E. coli, P. falciparum and D. radiodurans). Experimental results match known sequence data.
Topics: Algorithms; Bayes Theorem; Contig Mapping; DNA; Genome, Human; Humans; Models, Statistical; Reproducibility of Results; Restriction Mapping; Sensitivity and Specificity; Software
PubMed: 10786282
DOI: No ID Found -
Research in Virology 1990The DNA of 60 adenovirus type 8 (AV8) isolates (collected during the period 1961 to 1982, mostly in Western Germany) was analysed by 6 endonucleases and revealed 6...
The DNA of 60 adenovirus type 8 (AV8) isolates (collected during the period 1961 to 1982, mostly in Western Germany) was analysed by 6 endonucleases and revealed 6 different genome types, thus implying that the variability of AV8 is relatively low. It was found that 45 isolates belonged to the genome type D1. Restriction site maps of a prototype D1 and of all deviating restriction variants were elaborated for enzymes BamHI, BglII, Hind-III and SalI.
Topics: Adenoviridae; Cells, Cultured; DNA, Viral; Genetic Variation; Humans; Restriction Mapping
PubMed: 2087599
DOI: 10.1016/0923-2516(90)90034-g -
Journal of Computational Biology : a... 2000Optical mapping is a novel technique for determining the restriction sites on a DNA molecule by directly observing a number of partially digested copies of the molecule...
Optical mapping is a novel technique for determining the restriction sites on a DNA molecule by directly observing a number of partially digested copies of the molecule under a light microscope. The problem is complicated by uncertainty as to the orientation of the molecules and by erroneous detection of cuts. In this paper we study the problem of constructing a restriction map based on optical mapping data. We give several variants of a polynomial reconstruction algorithm, as well as an algorithm that is exponential in the number of cut sites, and hence is appropriate only for small number of cut sites. We give a simple probabilistic model for data generation and for the errors and prove probabilistic upper and lower bounds on the number of molecules needed by each algorithm in order to obtain a correct map, expressed as a function of the number of cut sites and the error parameters. To the best of our knowledge, this is the first probabilistic analysis of algorithms for the problem. We also provide experimental results confirming that our algorithms are highly effective on simulated data.
Topics: Algorithms; Biometry; DNA; Models, Statistical; Optics and Photonics; Restriction Mapping
PubMed: 10890404
DOI: 10.1089/10665270050081540 -
Genetics Oct 2007The wealth of sequence information available for Neurospora crassa and other fungi has greatly facilitated evolutionary and molecular analyses of this group. Although...
The wealth of sequence information available for Neurospora crassa and other fungi has greatly facilitated evolutionary and molecular analyses of this group. Although "reverse" genetics, in which genes are first identified by their sequence rather than by their mutant phenotypes, serves as a valuable new approach for elucidating biological processes, classical "forward" genetic analysis is still extremely useful. Unfortunately, mapping mutations and identifying the corresponding genes has typically been slow and laborious. To facilitate forward genetics in Neurospora, we have adapted microarray-based restriction-site-associated DNA (RAD) mapping for use with N. crassa oligonucleotide microarrays. This technique was used to simultaneously detect an unprecedented number of genomewide restriction site polymorphisms from two N. crassa strains: Mauriceville and Oak Ridge. Furthermore, RAD mapping was used to quickly map a previously unknown gene, defective in methylation-7 (dim-7).
Topics: DNA, Fungal; Genes, Fungal; Genetic Markers; Genome, Fungal; Microarray Analysis; Mutation; Neurospora crassa; Polymorphism, Genetic; Restriction Mapping
PubMed: 17660537
DOI: 10.1534/genetics.107.078147 -
Clinica Chimica Acta; International... Mar 2002The apolipoprotein E (apo E) polymorphism is associated with the risk of developing cardiovascular disease and the risk and the time of onset of Alzheimer's disease.... (Comparative Study)
Comparative Study
BACKGROUND
The apolipoprotein E (apo E) polymorphism is associated with the risk of developing cardiovascular disease and the risk and the time of onset of Alzheimer's disease. Therefore, the interest in apo E genotyping is high, both for epidemiological research and for the purpose of diagnosing dyslipidemia or dementia. The aim of our study was to compare and evaluate two different methods for apo E genotyping, both on the basis of polymerase chain reaction (PCR).
METHODS
Genomic DNA of 197 subjects was extracted from whole blood. The first method involved DNA amplification performing a PCR using specific primers and endonuclease restriction mapping. The second one was a DNA assay that used real-time PCR on the LightCycler instrument (Roche).
RESULTS
We obtained a 100% concordance between the two methods and we found a relative allelic frequency distribution typical for an Italian population.
CONCLUSIONS
The LightCycler (LC) allelic discrimination method for apo E genotyping seems to be rapid, simple and accurate, suggesting a possible successful use of this method for diagnostic purposes.
Topics: Alleles; Apolipoproteins E; Deoxyribonucleases, Type II Site-Specific; Humans; Polymerase Chain Reaction; Restriction Mapping
PubMed: 11814460
DOI: 10.1016/s0009-8981(01)00769-0