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Nucleic Acids Research Mar 1990
Topics: Bacteriophage lambda; Electromagnetic Fields; Electrophoresis; Electrophoresis, Agar Gel; Restriction Mapping
PubMed: 2138731
DOI: 10.1093/nar/18.5.1312 -
Methods in Molecular Medicine 2005The recognition of a homozygous deletion of genetic material in a tumor genome has been instrumental in several tumor suppressor gene searches. The representational... (Review)
Review
The recognition of a homozygous deletion of genetic material in a tumor genome has been instrumental in several tumor suppressor gene searches. The representational difference analysis (RDA) allows one to identify homozygous deletions even from among the high background of allelic losses that is typical for most cancers. RDA is a polymerase chain reaction (PCR)-based subtractive hybridization method. Two major obstacles to successful enrichment of target sequences from complex genomes were circumvented by RDA. Incomplete reassociation of complex DNA populations is overcome by using representative subpopulations of the tester and driver genomes. In addition, reiterated hybridization, selection, and amplification of the difference products introduces a kinetic component in the enrichment of target sequences. RDA thus enables the identification of homozygous deletions as small as 100 kilobases. Here, we provide a detailed protocol of the RDA procedure, including reflections on frequently encountered technical problems and on the particulars of its application in cancer.
Topics: Base Sequence; DNA; Genes, Tumor Suppressor; Homozygote; Humans; Nucleic Acid Hybridization; Polymerase Chain Reaction; Restriction Mapping; Sequence Deletion
PubMed: 15542904
DOI: 10.1385/1-59259-780-7:143 -
BioTechniques Jun 2004Plasmid construction by "forced" or "directional" ligation of fragments digested with two different restriction enzymes is highly efficient, except when inhibited... (Comparative Study)
Comparative Study
Plasmid construction by "forced" or "directional" ligation of fragments digested with two different restriction enzymes is highly efficient, except when inhibited digestion of one site favors vector recircularization. Such failures often result because incomplete double digestion is undetected in vector polylinkers or at terminal cloning sites on a PCR fragment. To test cleavage efficiency indirectly, a "monitor" plasmid is added to the digest. In a suitable monitor, the two test sites are separated by enough DNA (approximately 20% of full length) to distinguish the double digest from the failed single digest. To make this applicable to combinations of 32 popular cloning enzymes, we constructed a set of 4 monitors (pDM1, pDM2, pDM3, and pDM4). Each contains three polylinkers separated by stuffer segments of approximately 1 kb. The 32 sites are distributed in the polylinkers such that at least one plasmid in the set is diagnostic for each enzyme pair. The set is designed to be extended to up to 81 sites. A linearized version of the monitor allows for the determination of which of the two enzymes has failed in an incomplete double digest and is also useful when the target DNA is close to the size of the pDM backbone. The plasmids also serve as versatile self-monitoring cloning vectors for any site combination.
Topics: Cloning, Molecular; DNA Restriction-Modification Enzymes; Plasmids; Polymerase Chain Reaction; Restriction Mapping
PubMed: 15211749
DOI: 10.2144/04366ST03 -
BioTechniques Aug 2002A method was devised for generating nested deletions in DNA that exploits the difference in frequency of restriction sites recognized by compatible restriction...
A method was devised for generating nested deletions in DNA that exploits the difference in frequency of restriction sites recognized by compatible restriction endonucleases. A cloning vector was constructed that contains no common blunt-end or RsaI restriction sites and two 8-bp blunt-end restriction sites flanking a commodious multiple cloning site. DNA fragments are cloned into the multiple cloning site using blue-white selection, and nested deletions are generated by digesting the resulting plasmid with either SwaI or PmeI and partially digesting the insert DNA with RsaI. The DNAs are ligated and transformed, producing afamily of plasmids with different-sized deletions. The DNA sequence of these inserts can be rapidly determined, and the overlapping sequences can be assembled in silico to produce a large DNA contig. Nested deletions generated in this manner can also be used for the structure-function analysis of proteins.
Topics: Base Sequence; Cloning, Molecular; DNA Restriction Enzymes; DNA, Bacterial; Escherichia coli; Gene Deletion; Molecular Sequence Data; Restriction Mapping; Sequence Analysis, DNA; Sequence Deletion; Species Specificity
PubMed: 12188182
DOI: 10.2144/02332st02 -
Bioinformatics (Oxford, England) Sep 2006Cleaver is an application for identifying restriction endonuclease recognition sites that occur in some taxa but not in others. Differences in DNA fragment restriction...
UNLABELLED
Cleaver is an application for identifying restriction endonuclease recognition sites that occur in some taxa but not in others. Differences in DNA fragment restriction patterns among taxa are the basis for many diagnostic assays for taxonomic identification and are used in procedures for removing the DNA of some taxa from pools of DNA from mixed sources. Cleaver analyses restriction digestion of groups of orthologous DNA sequences simultaneously to allow identification of differences in restriction pattern among the fragments derived from different taxa.
AVAILABILITY
Cleaver is freely available without registration from its website (http://cleaver.sourceforge.net/) and can be copied, modified and re-distributed under the terms of the GNU general public licence version2 (http://www.gnu.org/licences/gpl). The program can be run as a script for computers that have Python 2.3 and necessary extra modules installed. This allows it to run on Gnu/Linux, Unix, MacOSX and Windows platforms. Stand-alone executable versions for Windows and MacOSX operating systems are available.
Topics: Algorithms; Binding Sites; Computer Graphics; DNA; DNA Restriction Enzymes; Protein Binding; Restriction Mapping; Sequence Analysis, DNA; Sequence Analysis, Protein; Software; Species Specificity; User-Computer Interface
PubMed: 16787976
DOI: 10.1093/bioinformatics/btl330 -
Genome Research Feb 1999Detailed restriction maps of microbial genomes are a valuable resource in genome sequencing studies but are toilsome to construct by contig construction of maps derived...
Detailed restriction maps of microbial genomes are a valuable resource in genome sequencing studies but are toilsome to construct by contig construction of maps derived from cloned DNA. Analysis of genomic DNA enables large stretches of the genome to be mapped and circumvents library construction and associated cloning artifacts. We used pulsed-field gel electrophoresis purified Plasmodium falciparum chromosome 2 DNA as the starting material for optical mapping, a system for making ordered restriction maps from ensembles of individual DNA molecules. DNA molecules were bound to derivatized glass surfaces, cleaved with NheI or BamHI, and imaged by digital fluorescence microscopy. Large pieces of the chromosome containing ordered DNA restriction fragments were mapped. Maps were assembled from 50 molecules producing an average contig depth of 15 molecules and high-resolution restriction maps covering the entire chromosome. Chromosome 2 was found to be 976 kb by optical mapping with NheI, and 946 kb with BamHI, which compares closely to the published size of 947 kb from large-scale sequencing. The maps were used to further verify assemblies from the plasmid library used for sequencing. Maps generated in silico from the sequence data were compared to the optical mapping data, and good correspondence was found. Such high-resolution restriction maps may become an indispensable resource for large-scale genome sequencing projects.
Topics: Animals; Chromosomes; DNA, Protozoan; Image Processing, Computer-Assisted; Microscopy, Fluorescence; Physical Chromosome Mapping; Plasmodium falciparum; Restriction Mapping
PubMed: 10022982
DOI: No ID Found -
Analytical Biochemistry May 1998Many new techniques in biomolecular chemistry and genomic analysis require the immobilization of molecular reagents on specially prepared surfaces. However, the process...
Many new techniques in biomolecular chemistry and genomic analysis require the immobilization of molecular reagents on specially prepared surfaces. However, the process of molecular fixation often interferes with or precludes the use of standard in vitro biochemical assays. Optical mapping is an emergent technology for genomic analysis which relies on the biochemical activity of DNA fixed to silanized glass surfaces. Optical mapping surfaces have been shown to be compatible with restriction endonucleases and a variety of DNA polymerases. The essential properties of biochemically active surfaces are poorly understood in most of the current technologies which utilize molecular fixation, including optical mapping. The purpose of this study is to use the powerful technique of atomic force microscopy, in combination with informative enzymatic assays, to correlate biochemical activity with microscopic surface structure. The results presented provide meaningful insight into the effect of surface preparation on the biochemical accessibility of surface-bound molecules. Novel analysis which may facilitate the automation of optical mapping is presented.
Topics: DNA; DNA Restriction Enzymes; Genetic Techniques; Hydrolysis; Indicators and Reagents; Kinetics; Microscopy, Atomic Force; Restriction Mapping
PubMed: 9606147
DOI: 10.1006/abio.1998.2640 -
BMC Genomics Jun 2008Alu repetitive elements are the abundant sequences in human genome. Diversity of DNA sequences of these elements makes difficult the construction of theoretical patterns...
BACKGROUND
Alu repetitive elements are the abundant sequences in human genome. Diversity of DNA sequences of these elements makes difficult the construction of theoretical patterns of Alu repeats cleavage by restriction endonucleases. We have proposed a method of restriction analysis of Alu repeats sequences in silico.
RESULTS
Simple software to analyze Alu repeats database has been suggested and Alu repeats digestion patterns for several restriction enzymes' recognition sites have been constructed. Restriction maps of Alu repeats cleavage for corresponding restriction enzymes have been calculated and plotted. Theoretical data have been compared with experimental results on DNA hydrolysis with restriction enzymes, which we obtained earlier.
CONCLUSION
Alu repeats digestions provide the main contribution to the patterns of human chromosomal DNA cleavage. This corresponds to the experimental data on total human DNA hydrolysis with restriction enzymes.
Topics: Alu Elements; DNA; DNA Restriction Enzymes; Genome, Human; Humans; Restriction Mapping; Sequence Alignment; Software
PubMed: 18578890
DOI: 10.1186/1471-2164-9-305 -
Virologica Sinica Oct 2016[Image: see text]
Pitfalls of restriction enzyme analysis in identifying, characterizing, typing, and naming viral pathogens in the era of whole genome data, as illustrated by HAdV type 55.
[Image: see text]
Topics: Adenovirus Infections, Human; Adenoviruses, Human; DNA, Viral; Genome, Viral; Humans; Phylogeny; Restriction Mapping; Virulence
PubMed: 27822718
DOI: 10.1007/s12250-016-3862-x -
Journal of Human Evolution Jan 1998Molecular phylogenetic relationships among all recognized species within the genus Macaca, were assessed using high-resolution restriction site mapping of the...
Molecular phylogenetic relationships among all recognized species within the genus Macaca, were assessed using high-resolution restriction site mapping of the mitochondrial ribosomal genes. By outgroup comparisons to other members of the cercopithecine subfamily, the macaques appear to be a monophyletic assemblage. Within the genus, the relationships are in general consistent with previous genetic studies, though they are less concordant with the separation of the species into four distinct species groups based on modification of the genitalia. Our data support: (1) Macaca sylvanus as sister clade to all Asian macaques; (2) the silenus group as a monophyletic assemblage, with the Sulawesi macaques diverging and colonizing Sulawesi much earlier than previously thought; (3) the fascicularis group as a paraphyletic assemblage, including all non-silenus group Asian macaques; (4) the sinica group as a monophyletic assemblage, possibly derived from a fascicularis-like ancestor; and (5) Macaca arctoides as a separate lineage from the sinica group, also originating from a fascicularis-like ancestor. This study supports the notion that species with more specialized genitalia evolved from less derived taxa, and in general are in agreement with the dispersal scenarios proposed by Fooden (1980) and Delson (1980) for the macaques.
Topics: Animals; Animals, Wild; Animals, Zoo; Binding Sites; DNA, Mitochondrial; DNA, Ribosomal; Humans; Macaca; Phylogeny; Restriction Mapping
PubMed: 9467779
DOI: 10.1006/jhev.1997.0171