Contour-clamped homogeneous electric field electrophoresis of Staphylococcus aureus

Contour-clamped homogeneous electric field (CHEF) electrophoresis is a technique of pulsed-field gel electrophoresis that enables the resolution of large fragments of DNA that cannot be resolved by conventional gel electrophoresis. The procedure involves the application of controlled electric fields that change direction at a predetermined angle to samples of DNA that have been embedded in an agarose gel matrix and digested with a restriction endonuclease. Adjustment of the electrophoresis conditions enables the separation of DNA fragments with lengths from 10 kilobases up to 9 megabases in a size-dependent manner in agarose gels. The banding patterns can be used for epidemiological typing, the separated DNA can be immobilized onto a membrane and used for genetic mapping, or individual fragments can be extracted and used for downstream genetic manipulations. The protocol requires specialized equipment and can be completed in a maximum of 7 days.     

A NheI macrorestriction map of the Neisseria meningitidis B1940 genome

Abstract A macrorestriction map of the Neisseria meningitidis strain B1940 genome was constructed by two-dimensional pulsed-field gel electrophoresis (2D-PFGE) techniques. Digestion of the genomic DNA with the restriction endonuclease NHe I revealed 15 fragments between 10 kb and 450 kb. The sum of the fragments and resolution of the linearized chromosome yielded a total genome size of about 2.3 Mbp. By overlapping methylation with the Alu I-methylase six Nhe I recognition sites could be blocked. Fragments were ordered by partial/complete 2D-PFGE of genomic DNA with and without prior Alu I methylation, respectively. All nine Alu I-methylase/ Nhe I and 14 Nhe I restriction sites could be mapped on a single circular chromosome. This map will serve as a useful tool for further genetic analysis of meningococci and exemplifies the power of non-radioactive 2D-PFGE techniques to construct large physical genome maps with a single restriction enzyme.     

Restriction mapping of recombinant lambda DNA molecules using pulsed field gel electrophoresis

We have integrated pulsed field gel electrophoresis with the partial digestion strategy of Smith and Birnstiel (1976, Nucleic Acids Res. 3,2387-2398) to generate a rapid and accurate method of restriction endonuclease mapping recombinant lambda DNA molecules. Use of pulsed field gels dramatically improves the accuracy of size determination and resolution of DNA restriction fragments relative to standard agarose gels. Briefly, DNA is partially digested with restriction enzymes to varying extents and then hybridized with a radiolabeled oligonucleotide which anneals specifically to one of the lambda cohesive (cos) ends, effectively end labeling only those digestion products containing that cos end. In this study, we have used an oligonucleotide hybridizing to the right cos end. DNA is then fractionated by pulsed field gel electrophoresis, the gel dried down, and cos end containing fragments visualized by autoradiography. Fragment sizes indicate the distances from the labeled cos end to each restriction site for the particular restriction enzyme employed. This procedure requires only minimal quantities of DNA and is applicable to all vectors utilizing lambda cos ends.     

A rapid two-dimensional fractionation of DNA restriction fragments

Genomic DNA that has been digested with a restriction endonuclease and fractionated by electrophoresis on an agarose gel can be recovered on glass-fiber filters by a new blotting scheme. The DNA fragments in each fraction are then digested with a second restriction nuclease and then separated on a slab gel, resulting in a two-dimensional display of the restriction fragments. This rapid fingerprinting technique is useful in the analysis of complex genomes and in the isolation and cloning of particular sequences.     

Organization of the yeast ribosomal RNA gene cluster via cloning and restriction analysis.

The restriction endonuclease EcoR1 cleaves Saccharomyces cerevisiae DNA, which codes for ribosomal RNA (rRNA), into seven fragments, A second restriction endonuclease, HindIII, cleaves the same yeast ribosomal DNA into two fragments. These two restriction enzymes each yield DNA segments that total about 5.9 megadaltons. The "repeat unit" of the yeast genes coding for rRNA is thus about 5.9 megadaltons or about 9000 base pairs long. The two HindIII-cleaved DNA fragments as well as one of the EcoR1-cleaved DNA fragments were purified and amplified by cloning in Escherichia coli. Three of the seven EcoR1-generated DNA fragments could then be ordered by treating the two cloned HindIII DNA fragments with EcoR1. This led the assignment of the two HindIII restriction sites. The various restriction DNA fragments were hybridized directly from the gel utilizing 32P-labeled 5 S, 5.8 S, 18 S, and 25 S rRNA. Identification of the various DNA restriction segments then led to the final ordering of the DNA fragments. The gene coding for the 5 S RNA is adjacent to the gene coding for the 35 S precursor rRNA. These two groups of genes thus occur as a cluster in the following sequence: [5 S-spacer]-[spacer-18 S-5.8 S-25 S-spacer]-[spacer-5 S]. The actual map of the DNA restriction fragments is presented.     

Rapid mapping of transposon insertion and deletion mutations in the large Ti-plasmids of Agrobacterium tumefaciens.

A procedure is presented, that has allowed the rapid assignment of transposon Tn1 and Tn7 insertion sites in the large (130 Md) nopaline Ti-plasmid pTiC58, to specific restriction enzyme fragments. Total bacterial DNA is isolated from Agrobacterium tumefaciens strain C58 mutants that carry a transposon in their Ti-plasmid, and digested with an appropriate restriction endonuclease. The fragments are separated on an agarose gel, denatured and transferred to nitrocellulose filters. These are hybridized against purified wild type pTiC58, or against segments of PTiC58, cloned in E. coli using pBR322 as a vector plasmid. DNA sequences homologous to the probe are detected by autoradiography, thus generating a restriction enzyme pattern of the plasmid from a digest of total bacterial DNA. Mutant fragments can be readily identified by their different position compared to a wild type reference. This protocol eliminates the need to separate the large plasmid from chromosomal DNA for every mutant. In principle, it can be applied to the restriction enzyme analysis of insertion or deletion mutants in any plasmid that has no extensive homology with the chromosome.     

Sequence specificity of DNA cleavage by Micrococcus luteus gamma endonuclease

DNA fragments of defined sequence have been used to determine the sites of cleavage by gamma-endonuclease activity in extracts prepared from Micrococcus luteus. End-labeled DNA restriction fragments of pBR322 DNA that had been irradiated under nitrogen in the presence of potassium iodide or t-butanol were treated with M. luteus gamma endonuclease and analyzed on high resolution, denaturing, polyacrylamide gels. Gamma endonuclease was found to cleave irradiated DNA preferentially at the positions of cytosines and thymines. DNA cleavage occurred immediately to the 3' side of pyrimidines in irradiated DNA and resulted in fragments that terminate in a 5'-phosphoryl group. These studies indicate that both altered cytosines and thymines may be important DNA lesions requiring repair after exposure to gamma radiation.     

A method for two-dimensional DNA electrophoresis (2D-DE): application to the immunoglobulin heavy chain variable region

The scarcity of single-copy probes creates difficulty in the generation of large-scale physical maps of mammalian gene families. A simple method of two-dimensional DNA electrophoresis (2D-DE) has been developed to overcome this problem. DNA (2 micrograms) is digested with a rare-cutting restriction endonuclease and size separated by pulsed-field gel electrophoresis (PFGE). The DNA, still contained within the lane of the PFGE gel, is digested with a second frequent-cutting restriction enzyme and is subjected to an electrical field perpendicular to that of the PFGE. 2D-DE allows the simultaneous mapping, to large restriction fragments, of all the genes detected by a particular probe. The human immunoglobulin variable region was used as an example for this procedure. Two VH5 genes, on 8- and 9-kb EcoRI fragments, were mapped to 200- and 65-kb SfiI fragments, respectively, by 2D-DE. This technique will be particularly useful in the generation of physical maps of complex human gene families and of repeat families.     

Specific arrangements of human satellite III DNA sequences in human chromosomes

DNA was extracted from various rodent-human somatic cell hybrids that contained single or a few human chromosomes. These DNAs were examined by a combination of restriction endonuclease digestion, gel electrophoresis, and filter hybridisation to radioactive satellite DNA probes following transfer of the denatured restriction fragments from a gel to a nitrocellulose filter. In this way the arrangement of sequences homologous to human satellite III were examined on human chromosomes 1, 7, 11, 15, 22 and X. It was found that the distribution of restriction endonuclease sites within satellite III DNA is different on different chromosomes.     

碳离子束辐照对质粒DNA的损伤效应

为了证明DNA双链断裂(DSB)片段分布与DNA序列有关的假设,采用32keV／μm的^12C^6 离子和45ke V／μm的^13C^6 离子分别辐照pUCl8质粒,结合限制性内切酶处理,进行琼脂糖凝胶电泳,分析DNA断裂和片段分布。结果表明,除了由一个DSB导致的线性DNA带外,还出现了一条新的、小分子量线性DNA带;限制性内切酶处理后,有另一条线性DNA带产生。证明重离子辐照诱导的DSB是非随机分布的,DNA分子上存在对电离辐射相对敏感的位点。     

A protocol for DNA fragment extraction from polyacrylamide gels

A simple and efficient method of purifying linear plasmid DNA from contaminating DNA fragments is described. Both vector and insert containing plasmids may be used without extensive purification, in particular without cesium chloride centrifugation. Careful deproteinization with phenol-chloroform allows efficient restriction enzyme digestion. Fragment separation can be performed immediately after restriction endonuclease digestion in a single 6% polyacrylamide gel. Extraction of DNA fragments from the gel is easy and gives a good yield. The DNA may be used for ligation and transformation without further purification.     

Specific Endonuclease R Fragments of Bacteriophage φX174 Deoxyribonucleic Acid

The restriction enzyme from Hemophilus influenzae, endonuclease R, cleaves phiX174 replicative-form deoxyribonucleic acid (DNA) into at least 13 specific limit fragments. The molecular weights of 12 of the fragments have been estimated by gel electrophoresis and electron microscopy. Using the genetic assay for small fragments of phiX DNA, we have shown that we can salvage markers from the endonuclease R phiX-RF fragments.     

Physical analysis and mapping of the Mycoplasma pneumoniae chromosome.

Field inversion gel electrophoresis was used for analysis of the chromosome of Mycoplasma pneumoniae. The restriction endonuclease SfiI (5'-GGCCNNNNNGGCC-3') generated 2 M. pneumoniae DNA fragments of approximately 437 and 357.5 kilobase pairs (kbp), whereas 13 restriction fragments ranging in size from 2.4 to 252.0 kbp resulted from digestion with ApaI (5'-GGGCCC-3'). Totaling the sizes of the individual restriction fragments from digestion with SfiI or ApaI yielded a genome size of 794.5 or 775.4 kbp, respectively. A physical map of the M. pneumoniae chromosome was constructed by using a combination of techniques that included analysis by sequential or partial restriction endonuclease digestions and use as hybridization probes of cloned M. pneumoniae DNA containing ApaI sites and hence overlapping adjacent ApaI fragments. Genetic loci for deoC, rrn, hmw3, and the P1 gene were identified by using cloned DNA to probe ApaI restriction fragment profiles.     

Extracellular nucleases of pseudomonas BAL 31. III. Use of the double-strand deoxyriboexonuclease activity as the basis of a convenient method for the mapping of fragments of DNA produced by cleavage with restriction enzymes.

We have previously characterized an extracellular nuclease from Pseudomonas BAL 31 which, in addition to other activities, displays a double-strand exonuclease activity which progressively shortens both strands of linear duplex DNA molecules from both termini. This degradation is accomplished without the introduction of detectable scissions away from the ends of the duplexes. When this nuclease is used to produce a series of progressively shortened samples from a linear duplex DNA, subsequent digestion of these samples with a site-specific restriction endonuclease and analysis of the resulting fragments by gel electrophoresis permits the rapid establishment of the order of the restriction enzyme fragments through the entire genome. This is accomplished by noting from the electropherograms the order in which the various restriction enzyme fragments become noticeably shortened or disappear. Using this method, the five cleavage sites for the endonuclease Hpa I and the single cleavage sites for the nucleases Hpa II and Pst I have been mapped in PM2 bacteriophage DNA. In a more stringent test of the method, 18 of the 24 fragments produced by cleavage of coliphage lambdab2b5c DNA with the Pst I nuclease have been mapped, and five of the six remaining fragments have been assigned to small regions of the genome.     

Physical map of the bacteriophage T5 genome based on the cleavage products of the restriction endonucleases SalI, SmaI, BamI, and HpaI.

A physical map of the bacteriophage T5 genome was constructed by ordering the fragments produced by cleavage of T5 DNA with the restriction endonucleases SalI (4 fragments), SmaI (4 fragments), BamI (5 fragments), and HpaI (28 fragments). The following techniques were used to order the fragments. (i) Digestion of DNA from T5 heat-stable deletion mutants was used to identify fragments located in the deletable region. (ii) Fragments near the ends of the T5 DNA molecule were located by treating T5 DNA with lambda exonuclease before restriction endonuclease cleavage. (iii) Fragments spanning other restriction endonuclease cleavage sites were identified by combined digestion of T5 DNA with two restriction endonucleases. (iv) The general location of some fragments was determined by isolating individual restriction fragments from agarose gels and redigesting the isolated fragments with a second restriction enzyme. (v) Treatment of restriction digests with lambda exonuclease before digestion with a second restriction enzyme was used to identify fragments near, but not spanning, restriction cleavage sites. (vi) Exonucleases III treatment of T5 DNA before restriction endonuclease cleavage was used to locate fragments spanning or near the natural T5 single-chain interruptions. (vii) Analysis of the products of incomplete restriction endonuclease cleavage was used to identify adjacent fragments.     

Detection of DNA fragmentation and endonucleases in apoptosis

DNA degradation during apoptosis is endonuclease mediated and proceeds through an ordered series of stages commencing with the production of large DNA pieces of 300 kb which are then degraded to fragments of 50 kb. The 50-kb fragments are further degraded, in some but not all cells, to smaller pieces (10-40 kb) releasing the small oligonucleosome fragments that are detected as a characteristic DNA ladder on conventional agarose gels. Methodology is presented for the detection of both DNA ladders and the initial stages of DNA fragmentation using pulsed-field gel electrophoresis. We have developed electrophoresis conditions that resolve large fragments of DNA and also retain the smaller fragments on the same gel. Methods for the detection of endonuclease activities responsible for the cleavage of DNA during apoptosis are also presented.     

应用Agilent 2100 Bioanalyzer分析限制性显示技术制备的HIV片段库

使用Agilent 2 10 0Bioanalyzer分析限制性显示技术 (restrictiondisplay ,RD)制备的HIV片段库 .利用合适的限制酶从质粒上获得HIVB亚型代表株U2 6 94 2全基因cDNA ,然后将目的片段进行Sau3AⅠ消化 ,在消化得到的片段两端加接头 ,利用通用引物进行PCR扩增 ,扩增结果通过琼脂糖凝胶电泳以及Agilent 2 10 0Bioanalyzer两种方法分析 .结果显示 ,Agilent 2 10 0Bioanalyzer比琼脂糖凝胶电泳能更快速、直接和客观地反映RD技术制备的DNA片段的大小以及含量 ,并能对RD PCR过程中片段自身连接以及优势扩增的现象进行直接的监控作用 .     

Controlled partial restriction digestions of DNA by competition with modification methyltransferases

Competitive reactions, using defined ratios of DNA restriction methyltransferase to endonuclease, are shown to result in reliable partial restriction digests of DNA. This method is suitable over a wide range of DNA concentrations and works on DNA in liquid or embedded in agarose. Simultaneous methylase/endonuclease reactions using endonucleases that cleave human DNA very infrequently, such as ClaI or NotI, should generate very large discrete partial DNA fragments suitable for physical mapping in the million base-pair range. Another possible application of methylase/endonuclease competitive reactions is the production of defined partial digests for making cosmid, lambda, or other genomic libraries.     

DNA polymorphism in strains of the genus Brucella

Preparations of DNA from 23 Brucella strains including 19 reference strains were compared by restriction endonuclease analysis. Pulsed-field gel electrophoresis resulted in optimal resolution of fragments generated by digestion with low-cleavage-frequency restriction enzymes such as XbaI. By this technique, five electrophoretypes were distinguished in five reference strains of the different species, i.e., B. abortus, B. melitensis, B. suis, B. canis, and B. ovis. Minor profile differences allowed us to discriminate between most biovars within a species. However, the differences in the DNA patterns of different field strains of biovar 2 of B. melitensis were not sufficient to serve as markers for epidemiological studies. From the XbaI fragments, we were able to estimate the size of the genomes of B. abortus 544T and B. melitensis 16 MT. This method revealed a relationship between DNA fingerprints, species, and pathovars which could shed light on problems concerning the classification and evolution of members of the genus Brucella.     

Comparison of the fine structure of mitochondrial DNA from Saccharomyces cerevisiae and S. carlsbergensis: electron microscopy of partially denatured molecules.

Denaturation-maps of mitochondrial DNA from Saccharomyces cerevisiae and S. carlsbergensis have been derived from electron microscopic observations of partially denatured complete circular molecules and large fragments of these circles. The mitochondrial DNA from the two species differ by 6% in total length, but seems from the maps to contain some regions of apparent close homology. The cleavage pattern of the two DNAs by the restriction endonuclease EcoRI is compared by gel electrophoresis.