Mapping the order of DNA restriction fragments

A straightforward method was designed for mapping the order of DNA restriction fragments obtained by a double and two single digestions, without the necessity of using a computer or a radioactive label. All possible solutions compatible with a pre-set level of error in the determination of sequence lengths are obtained. The primary assumptions are given, and the appropriate modifications of the algorithm are presented as a function of any assumptions one is unable (or unwilling) to make. Use of the method in connection with end-labeled fragments is also described.     

Antibiotic induced electrophoretic mobility shifts of DNA restriction fragments.

Several antibiotics, netropsin, distamycin A, actinomycin D, Hoechst 33258 and olivomycin, which demonstrate base specificity in their DNA binding properties have been found to alter the electrophoretic mobility of DNA restriction fragments in native polyacrylamide gels. The antibiotics mostly reduced the migration of larger DNA fragments, but netropsin and Hoechst 33258 were observed to increase the migration rate of several DNA fragments of intermediate size. DNA fragments of similar molecular weight which comigrate as a single gel band can at times be separated as the result of differential mobility shifts promoted by antibiotic DNA complex formations.     

Multiplex random priming of internal restriction fragments for DNA sequencing.

An approach for DNA sequencing is described that circumvents the need for synthetic oligonucleotide primers, which seriously restrict the progress of DNA sequencing in the commonly used protocol. The method is based on the use of short restriction fragments as primers randomly distributed along single-stranded templates. Premapping of target DNA is eliminated and subcloning manipulation is minimized. This method has been used successfully for sequencing genes in the range of 2 kb, for which about 10 restriction fragment primers per kilobase were sufficient to generate a continuous overlapping sequence in alignment. The approach has also been readily applied for an automated sequencing system with the fluorescent chain-terminating dideoxynucleotides, thus implying its potential for sequencing large genomic DNAs.     

Electrophoresis of DNA restriction fragments in poly-N-acryloyl-tris gels

Poly-N-acryloyl-tris(hydroxymethyl)aminomethane (NAT) gels were evaluated as a matrix for DNA electrophoresis. The resolution of DNA restriction fragments in three poly(NAT)-N,N'-methylenebisacrylamide (Bis) gels (4, 5, and 6%) was compared with the resolution in polyacrylamide (AA)-Bis gels of the same percentage. Poly(NAT) gels were found to give a substantially improved separation of DNA fragments larger than 200 bp. In contrast to poly(AA) gels, DNA fragments of up to 4 kbp were well resolved in the new matrix. By pulse-field electrophoresis the useful separation range of poly(NAT) gels was expanded to at least 23 kbp. For DNA fragments below 10 kbp, the resolution was better than that in a 0.7% agarose gel. Thus poly(NAT) gels are most suitable for the electrophoretic separation of DNA molecules whose size is out of the optimal fractionation range of poly(AA) or agarose gels.     

Detection of herpes simplex virus type-2 DNA restriction fragments in human cervical carcinoma tissue.

DNA extracted from eight human cervical carcinomas, one lymph node metastasis and related control tissue was examined for the presence of herpes simplex virus (HSV) DNA sequences. Southern blot transfers of tumour and control DNA were hybridised with radioactively labelled cloned probes representing 70% of the HSV-2 genome. Specific hybridisation to HSV DNA sequences was observed in one of eight carcinoma tissues analysed. Hybridisation of HSV-2 DNA probes to BamHI and XhoI restriction enzyme fragments of tumour cell DNA which co-migrated with authentic HSV-2 viral fragments identified co-linear HSV-2 DNA sequences comprising 3% of the HSV-2 genome, between map coordinates 0.582 and 0.612. The remaining eight tumour and all control tissues analysed, showed no specific hybridisation to any of the probes used at levels of sensitivity which would detect 0.5 copies/cell of HSV-2 DNA restriction fragments of 2 kb or greater.     

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.     

Cloning of restriction fragments of DNA from staphylococcal bacteriophage phi 11.

EcoRI fragments of Staphylococcus aureus bacteriophage phi 11 DNA were cloned in vector plasmid pSA2100 in S. aureus. The clones were analyzed in marker rescue experiments with suppressor- and temperature-sensitive mutants of phi 11 to correlate the genetic and physical map. Several mutants could be identified on the physical map, and a clone containing fragment EcoRI-B of phi 11 DNA expressed immunity to phage infection. In addition, it was found that recombinant plasmids containing phi 11 DNA sequences can be transferred by high-frequency transduction after phage phi 11 infection of host cells.     

Cloning DNA restriction endonuclease fragments with protruding single-stranded ends

A new method of in vitro recombination was employed to construct plasmids containing lac promoter fragments 64 bp and 144 bp long. The 64 bp HpaII-HhaI fragment contains the binding site for the catabolite activator protein (CAP). The HpaII-HaeIII 144 bp fragment includes the binding sites for RNA polymerase, the lac repressor and CAP. The method utilizes the ability of T4 DNA polymerase to make flush-ended DNA either by filling in a recessed 3'-end or by exonucleolytic removal of a protruding 3'-end. The treated fragments were then blunt-end ligated to the filled-in EcoRI cloning sites of the plasmids pVH51 and pBR322 using T4 ligase. In this process, the EcoRI sites were regenerated on the fragment ends thus facilitating the subsequent isolation of the fragments from their cloning vectors.     

Multi-priming sequencing: a DNA sequencing method involving restriction enzyme-digested DNA fragments as primers.

An improved strategy for fluorescence-labeled dideoxy chain termination sequencing involving restriction enzyme-digested DNA fragments as primers, which are prepared from the DNA to be sequenced, is described. By using modified nucleoside triphosphates for strand protection in chain termination reactions, newly synthesized chains were detached from a primer at the regenerated recognition site by means of suitable restriction enzyme digestion. The digests could be analyzed with commercial automated DNA sequencers. Thus, by using restriction DNA fragments (double-stranded) as primers, sequence information was obtained from both "minus" and "plus" single-stranded DNA templates without subcloning. Nor is the synthesis of oligonucleotide primers needed. This method, named "Multi-Priming Sequencing," was proven to be time-saving, economical, and effective compared to conventional methods.     

Cloning and characterization of restriction fragments of phage Mu DNA

Abstract The Eco RI-A and B, the Bam HI-C and the two Eco RI- Bam HI restriction fragments of transposing phage Mu DNA were inserted into vector plasmids pRSF2124 and pBR322. Quantitative marker rescue experiments for five genes located on the Eco RI-A fragment revealed complementation of phages carrying amber mutations in genes C , E , H , F and L . The in vitro coding capacity of the recombinant plasmids was assayed in a DNA-directed protein synthesizing system.     

Reverse-phase HPLC of DNA restriction fragments and ribooligonucleotides on uncoated Kel-F powder.

Uncoated Kel-F powder offers some unique features as a support for reverse-phase HPLC of oligonucleotides and DNA restriction fragments. Compounds are eluted from the column by a gradient of acetonitrile (0 tto 18% v/v) in 0.1 M aqueous triethylammonium acetate. In contrast to RPC-5 chromatography, oligonucleotides are not eluted by aqueous salt solutions alone, and the separation of restriction fragments depends only on the chainlength. The packing material is cheap, easy to pack, chemically inert, and does not bleed, so that separations are highly reproducible. The DNA loading capacity for Kel-F is presently inferior to RPC-5, but recovery of microgram amounts of material is typically better than 50%.     

Ligation of restriction endonuclease-generated DNA fragments using immobilized T4 DNA ligase

Phosphorylation of terminal deoxynucleotidyl transferase within leukemic cells has been demonstrated, using ³²P labelling of intact cells in culture, followed by immunoprecipitation of the cellular extracts using an anti-terminal transferase antiserum. The phosphate linkage was found to involve serine and threonine residues. Purified calf thymus terminal transferase served as a substrate for cyclic AMP independent protein kinase obtained from leukemic cells. Phosphorylation $\text{in vitro}$ of terminal transferase was accompanied by increased activity and decreased inhibition by excess ribo-ATP. These results indicate that terminal transferase is a physiologic cyclic AMP independent protein kinase substrate, and that this reaction may be important in its control.     

A sizing artifact of DNA restriction fragments with unusually long single-stranded termini.

The restriction endonucleases (ENases) BstNI (CCATGG) and EcoRII (CCATGG) both cleave DNA at the same time sequences, but only EcoRII produces 5-nucleotide (nt) cohesive ends and is inhibited by 5-methylation of the inner cytosine. The low-Mr fragments in digests of mouse DNA made with these two ENases exhibit different mobilities during agarose-gel electrophoresis. The difference in the mobilities of the BstNI and EcoRII fragments from mouse DNA was not due to closely spaced, differentially methylated sites, or to alternate mechanisms such as circularization of the long cohesive ends of the EcoRII fragments, or to residual bound protein. Rather, it was due to the unusually long 5-nt single-stranded (ss) ends of fragments produced by EcoRII digestion, since the slower mobility of the EcoRII fragments was abolished by treatment with ss-specific nuclease. Similar mobility differences between BstNI and EcoRII fragments which could be removed by ss nuclease were also observed in digests of simian virus 40 DNA.     

A method which facilitates the ordering of DNA restriction fragments

Summary The Southern transfer technique has been used to provide a generally applicable method for ordering DNA restriction fragments. It involves electrophoresis of partially digested DNA, transfer to nitrocellulose filter paper and annealing to a ³²P-labelled fragment. Only those partials containing that particular fragment will reanneal to the probe and produce bands on autoradiography. The size of each partial in the labelled set is the sum of the size of the fragment used as probe and of one or more adjacent fragments. Thus the size of the adjacent fragments can be determined from the size increments of this set of partials. The method is illustrated by the mapping of certain BamHI sites on coliphage 186 DNA.     

Interaction of the MvaI restriction enzyme with synthetic DNA fragments

The cleavage of synthetic DNA duplexes by the restriction endonuclease MvaI has been studied. The main result of the cleavage experiments is that MvaI cleaves unmodified duplexes in two single strand scissions in separate events and that the two strands are cleaved at significantly different rates. One strand nicks within the recognition site do not affect the cleavage. Furthermore, neither a pyrophosphate internucleotide bond modification in one strand nor the absence of one phosphate group at the central dA-residue of the recognition site do inhibit the cleavage of the second strand.