Separation of DNA fragments by high-resolution ion-exchange chromatography on a nonporous QA column

A nonporous QA column (a strong anion exchanger) was used for HPLC of DNA fragments. This column was successfully employed to separate small (ca. 10 bp) and intermediate size (ca. 10 kbp) DNA fragments from each other. The column also separated double-stranded DNA from its single-stranded form, and circular DNA molecules from linear ones. The entire separation process was completed within 60 min. The recovery of DNA fragments in each run was above 95%. High resolution was obtained both at an analytical level (microgram scale) and at a preparative level (100 micrograms scale). In view of time efficiency, recovery, and resolution, the nonporous QA column is superior to other porous ion-exchange columns and expected to be a very useful tool in molecular biological studies.     

Preparative fractionation of DNA restriction fragments by high pressure column chromatography on RPC-5.

High pressure liquid chromatography on the RPC-5 reversed-phase ion exchange system has been shown to have several potential applications as an initial high capacity step in the isolation of specific DNA restriction fragments. The fractionation of the Hinc II digest of lambda DNA, which contains 35 fragments with "flush ends" ranging in size from 3 x 10(6) to 7 x 10(4) daltons, has been used as a model system. Under certain conditions there are some restriction fragments whose elution relative to other fragments is different on RPC-5 chromatography than it is on gel electrophoresis. In some special circumstances it is possible to obtain satisfactory yields (60-70%) of a pure restriction fragment after a single passage through an RPC-5 column.     

Method for the characterization of size-exclusion chromatography media for preparative purification of DNA restriction fragments

A protocol has been developed to characterize the potential of size exclusion chromatography media for the separation of DNA fragments at a preparative scale. A standard DNA mixture composed of -DNA cut with the restriction enzymes, AluI and HaeIII, was chromatographed on a column packed with the gel of interest. Fractions were collected and examined by PAGE. A digitized image of the gel was analyzed with the aid of a computer software program to determine the DNA fractionation range and selectivity curve for each chromatography gel. Four gels were characterized using this protocol. The effect of the elution buffer ionic strength on the fractionation of DNA was also investigated.The US Government right to retain a non-exclusive royalty-freelicense in and to any copyright is acknowledged.     

Influence of A-T content on the fractionation of DNA restriction fragments by RPC-5 column chromatography.

The properties of the fractionated Hae III fragments of pRZ2 DNA (Patient, R.K., Hardies, S.C., and Wells, R.D. (1979) J. Biol. Chem. 254, 5542-5547) were studied in an effort to determine why several of the fragments bind more tightly to RPC-5 than expected on the basis of their length. The purified fragments were analyzed for their nucleotide composition by direct determination of their constituent mononucleotides and by analytical CsCl and Cs2SO4 density gradient analyses. A-T-rich fragments elute at higher salt concentrations than fragments of equivalent size which are not A-T-rich. In addition, denaturation mapping studies by electron microscopy indicate that an A-T-rich run within an otherwise G-C-rich fragment can give rise to delayed elution. At least one other factor influences the separation of DNA restriction fragments by RPC-5 chromatography. Some of the fragments in this digest which elute later than predicted from their size either contain known genetic regulatory sites or bind regulatory proteins.     

Separation of DNA restriction fragments by ion-exchange chromatography on FPLC columns Mono P and Mono Q

Separation of DNA restriction fragments by FPLC ion-exchange chromatography on Mono Q and Mono P columns was investigated. The columns were found to be particularly suitable for the separation of fragments up to 500–600 bp long. Larger fragments can also be separated although less effectively. We found the following practical working ranges for the parameters investigated: pH, 4 to 11; flow rate, 0.05 to 0.6 ml/min corresponding to separation times between 2 and 20 h. (better resolution is achieved at lower flow rates); gradient slope; between 0.5 mm eluting salt/ml buffer and over 5 mm/ml (better resolution is achieved at lower gradient slopes; eluting ionic strength was found to be independent of gradient slope); gradient composition, chloride salts of smaller monovalent cations eluted the DNA at lower ionic strengths but separations obtained were similar; additives, substances such as urea, formamide, and EDTA can be added without chromatographic effects; sample amount: amounts from 2.5 to 200 μg were applied, corresponding to single peak content of from 42 ng to 74μg DNA. Yields were generally over 90% and the chromatographed DNA was fully accessible to restriction enzyme cleavage. Separations occurred predominantly according to DNA size, but AT-rich fragments were retarded in a predictable way.     

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.     

Repeating restriction fragments of human DNA.

Human DNA digested with Hae III showed multiple repeats of a 170 base pair fragment. The most prominent band was the 340 base pair dimer, estimated to be 0.8% of the entire genome. Eco R1 and Hha I yielded fragments with similar electrophoretic mobility to the Hae III dimer. In each case this band was markedly enriched in DNA reassociating at a 0t of less than or equal to 1. Hybridization of the Hae III dimer to gels eluted on to filters demonstrated that the multiple Hae III fragments and Eco R1 fragments contained compatible sequences. These sequences may comprise a distinct subclass of DNA.     

Two methods to detect DNA fragments produced by restriction enzymes

This report summarizes two methods for detecting limited amounts of DNA from restriction endonuclease digests. The first is a photographic system for visualizing ethidium bromide-stained DNA fragments in agarose gels which can detect as little as 50-100 pg DNA per band. The second technique is direct sulfonation of DNA fragments bound to nylon membranes followed by visualization of the fragments by nonradioactive immunoblot methods. The immunohistochemical staining can detect 10 pg DNA per band. The direct sulfonation technique is not intended to identify specific DNA sequences; DNA-DNA hybridization with sulfonated probes has previously been described (P. Lebacq, D. Squalli, M. Duchenne, P. Poulety, and M. Johannes (1988) J. Biochem. Biophys. Methods 15, 255-266). Direct sulfonation can be used when samples are relatively free of contaminating nucleic acids and is a useful alternative to end-labeling. These highly sensitive techniques may be suitable when the DNA source is of limited quantity or in instances where radiolabeling is not permitted.     

Hydrophobic behavior of sciatin and transferrin on a size-exclusion column

Sciatin and transferrin are very similar glycoproteins which differ slightly in their carbohydrate content. In two-dimensional gel electrophoresis, they have one different isoform at pI lower than 5.77. However, highly different elution profiles have been recorded following size-exclusion HPLC. The use of a I-125 silica gel column has thus provided convincing evidence that both proteins do not show exactly the same hydrophobic three-dimensional structure.     

A bacteriophage lambda vector for cloning large DNA fragments made with several restriction enzymes

Lambda derivatives are described that can be used for cloning DNA fragments of about 20 kilobase pairs (kb) generated by restriction enzymes EcoRi, HindIII, BamHI, MboI and BglII. Recombinants can be selected by their Spi- phenotype and their propagation is facilitated by the presence of a chi site.     

Long-range guanine oxidation in DNA restriction fragments by a triplex-directed naphthalene diimide intercalator

Naphthalene diimide (NDI), a powerful oxidant that binds avidly to DNA by intercalation, is seen to damage the 5' guanine of 5'-GG-3' sites by photoactivated charge transport through DNA. When covalently tethered to the center of a triplex-forming oligonucleotide and delivered by triplex formation within a pyrimidine.purine-pyrimidine motif to a specific site on a restriction fragment, NDI can photooxidize guanine over at least 25-38 bp in each direction from the site of binding. Charge migration occurs in both directions from the NDI intercalator and on both DNA strands of the target, but the oxidation is significantly more efficient to the 3' side of the triplex. NDI and octahedral rhodium intercalators, when tethered directly to the 5' terminus of the triplex-forming strand as opposed to the center, generate significant amounts of oxidative damage only in the immediate vicinity of the intercalation site. Given that long-range charge transport depends on DNA stacking, these results suggest that the base stack is distorted at the 5' end of the triplex region in the duplex-triplex junction. Targeting of photooxidative damage by triplex formation extends our previous studies of long-range charge transport to significantly longer DNA sequences through a strategy that does not require covalent attachment of the photooxidant to the DNA being probed. Moreover, triplex targeting of oxidative damage provides for the first time a typical distance distribution for genomic charge transport of approximately 200 A around the oxidant.     

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.     

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.     

Preparation of megabase-sized tomato DNA and separation of large restriction fragments by field inversion gel electrophoresis (FIGE)

The Schwartz and Cantor technique for releasing and fractionating megabase-sized DNA from agarose-embedded cells is beginning to bridge the gap in resoluation between classical genetics and current molecular DNA techniques, particularly in mammalian systems. As yet no conditions have been described for preparing plant DNA that is of sufficient length to allow similar long-range restriction mapping experiments in plant systems. In this report, we describe the application of the Schwartz and Cantor technique for preparing high molecular weight DNA from embedded tomato leaf protoplasts, as well as conditions for generating and fractionating large restriction fragments, by field inversion gel electrophoresis (FIGE). The bulk of DNA released from lysed protoplasts was at least 2 Mb in size and amenable to restriction digestion as shown by hybridizing Southern blots with, among others, a probe for the Adh-2 gene of tomato. Restriction fragments as large as 700 kb were detected. Chloroplast DNA is isolated intact, amenable to restriction analysis and, in its native form, not mobile in FIGE.     

Fractionation of simian virus 40 DNA fragments by RPC-5 column chromatography

This report describes several modifications of the original radioenzymatic assay for serotonin (4) which increase the sensitivity of the assay 10-fold as well as enhance its reliability. Serotonin is converted to [³H]melatonin, in two steps. First, serotonin is acetylated to N-acetylserotonin by acetic anhydride. The N-acetylserotonin is then incubated with hydroxyindole-O-methyltransferase and S-[methyl-³H]adenosyl methionine and is converted to [³H]melatonin. The radioactive melatonin is extracted with toluene-isoamyl alcohol (7:3), dried, reconstituted, isolated by one-dimensional, silica gel, thin-layer chromatography, and counted in a liquid scintillation counter. The assay is specific and sensitive to approximately 5 pg of serotonin and thus can be used to measure serotonin levels in single brain nuclei or microliter quantities of biological fluids. The assay can be easily adapted for the direct measurement of N-acetylserotonin. A large number of samples can be assayed in a single working day.     

Terminal fragments of herpes simplex virus DNA produced by restriction endonuclease.

Digestion of HSV-1 DNA with λ 5′-exonuclease prior to digesting the DNA with the $\text{Eco R}$ I restriction endonuclease specifically affects two of the fragments normally obtained after restriction endonuclease digestion. Therefore these two fragments contain the sequences which occur at the termini of HSV-1 DNA. One of the fragments affected is a “minor” fragment which is always present in less than molar yield. The possible relationship between the occurrence of minor $\text{Eco R}$ I fragments and the partial refractoriness of HSV-1 DNA to λ 5′-exonuclease digestion is discussed.