The derivation of a restriction endonuclease map forAlcelaphine herpesvirus 1 DNA
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The ordering of restriction endonuclease fragments of HSV-2 DNA for physical maps has been studied using molecular hybridization techniques and the cleavage of isolated restriction endonuclease fragments with further restriction endonucleases. Physical maps for the fragments produced by EcoRI, Hind III, Bgl II, Xba and Hpa I have been constructed. The mol. wt. of the various regions which constitute HSV-2 genome are very similar to the corresponding mol. wt. in the HSV-1 genome.
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Genomic DNA of the myxobacterium Myxococcus xanthus was digested with the rare cutting restriction endonuclease AseI or SpeI, and the restriction products were separated by pulsed-field gel electrophoresis. Transposons Tn5-132 and Tn5 lac, which contain AseI restriction sites, were used to determine the number of restriction fragments in each band. The size of the genome was determined by adding the molecular sizes of the restriction products. The genomes of strains DK101, MD2, and DZF1 have identical restriction patterns and were estimated to be 9,454 +/- 101 kilobase pairs from the AseI digestions and 9,453 +/- 106 kilobase pairs from the SpeI digestions. DK1622, which was derived from DK101 by treatment with UV light, has suffered a 220- to 222-kilobase-pair deletion that removed an AseI and an SpeI restriction site. The deleted DNA may consist exclusively of Mx alpha-associated sequences.
Myxococcus xanthus
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A computer program is described which constructs maps of restriction endonuclease cleavage sites in DNA molecules, given only the fragment lengths. The program utilizes fragment length data from single and double restriction enzyme digests to generate maps for linear or circular molecules. The search for a map can be limited to the unknown (insert) region of a recombinant phage or plasmid. Typical restriction maps with four or five enzymes which cut at three to five unknown sites can be calculated in a few minutes.
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Restriction endonucleases are indispensable tools in molecular biology and biotechnology.Type II restriction endonucleases are part of restriction modification systems.DNA fragment extraction and restriction mapping are the basis for several biotechnological activities.WebFARM is a server application for identifying restriction endonuclease recognition sites and to give information regarding restriction mapping for given nucleotide sequences.WebFARM analyses given nucleotide sequence and identify restriction site for selected restriction endonucleases.It will also provide frequency of restriction for each restriction endonuclease.
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A computer program is described which allows for the manipulation of restriction maps of various DNA fragments to demonstrate techniques used in DNA cloning and to predict and/or confirm experimental results. This program is capable of reading in restriction enzyme cleavage sites for several different DNA molecules of interest. This information is then compiled in order to form restriction maps which can then be processed by digestion with restriction endonucleases and treatment with other common DNA modifying enzymes. Ligation can then be simulated by joining fragments with complementary ends in all possible orientations, producing restriction maps of the products. The resulting recombinants can then be further analyzed by physical mapping with appropriate restriction endonucleases. This program was written in Pascal on an Apple II computer.
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In vitro recombination
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Quantitative DNA fiber mapping (QDFM) is a high-resolution technique for physical mapping of DNA. The method is based on hybridization of fluorescently labeled DNA probes to individual DNA molecules stretched on a chemically modified glass surface. We now demonstrate and validate a rapid QDFM-based approach for the mapping of multiple restriction sites and precise localization of restriction fragments in large genomic clones. Restriction fragments of a 70-kb P1 clone (P1-70) containing the 5’ region of the human apolipoprotein B gene (APOB) were subcloned and mapped along straightened P1-70 DNA molecules. Multicolor fluorescence in situ hybridization (FISH) and digital image analysis allowed us to rapidly position 29 restriction fragments, ranging in size from 0.5 kb to 8 kb, and to map 43 restriction sites. The restriction map obtained by QDFM was in excellent agreement with information obtained by RecA-assisted restriction endonuclease (RARE) cleavage, long-range PCR, and DNA sequence analyses of the P1-70 clone. These data demonstrate that QDFM is a rapid, reliable method for detailed restriction site-mapping of large DNA clones.
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genomic DNA
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Southern blot
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