Two-dimensional strandness-dependent electrophoresis: a method to characterize single-stranded DNA, double-stranded DNA, and RNA-DNA hybrids in complex samples

We describe two-dimensional strandness-dependent electrophoresis (2D-SDE) for quantification and length distribution analysis of single-stranded (ss) DNA fragments, double-stranded (ds) DNA fragments, RNA-DNA hybrids, and nicked DNA fragments in complex samples. In the first dimension nucleic acid molecules are separated based on strandness and length in the presence of 7 M urea. After the first-dimension electrophoresis all nucleic acid fragments are heat denatured in the gel. During the second-dimension electrophoresis all nucleic acid fragments are single-stranded and migrate according to length. 2D-SDE takes about 90 min and requires only basic skills and equipment. We show that 2D-SDE has many applications in analyzing complex nucleic acid samples including (1) estimation of renaturation efficiency and kinetics, (2) monitoring cDNA synthesis, (3) detection of nicked DNA fragments, and (4) estimation of quality and in vitro damage of nucleic acid samples. Results from 2D-SDE should be useful to validate techniques such as complex polymerase chain reaction, subtractive hybridization, cDNA synthesis, cDNA normalization, and microarray analysis. 2D-SDE could also be used, e.g., to characterize biological nucleic acid samples. Information obtained with 2D-SDE cannot be readily obtained with other methods. 2D-SDE can be used for preparative isolation of ssDNA fragments, dsDNA fragments, and RNA-DNA hybrids.     

Two-dimensional (2D) DNA crystals assembled from two DNA strands

Sequence symmetry has been used to simplify the design of a DNA double-crossover (DX) molecule. The resulting DX molecule can self-assemble into two-dimensional (2D) crystalline arrays, but only requires two instead of otherwise four different DNA strands.     

Rapid and quantitative recovery of DNA fragments from gels by displacement electrophoresis (isotachophoresis)

The use of displacement electrophoresis (synonymous to isotachophoresis, steady-state stacking, and moving boundary electrophoresis) for recovery of DNA fragments from agarose and polyacrylamide gels is described. Complete recovery of DNA molecules ranging from oligonucleotides to 20 000-basepairs-long fragments was achieved. The DNA is recovered in a small volume (0.1-0.3 ml) and can be used directly in enzyme-mediated cleavage and ligation reactions. The recovered DNA contained no inhibitory contaminants as revealed by ligation or restriction enzyme cleavage.     

Two-dimensional electrophoresis of peptides from human-CHO cell hybrids containing human chromosome 21

Peptide expression influenced by human chromosome 21 was examined by comparing two-dimensional electrophoretograms of a human-hamster hybrid cell containing human chromosome 21 with its parent hamster cell and a revertant of the hybrid which had segregated the chromosome 21 genes for SOD-1, GARS, and a cytotoxic cell-surface antigen. Certain peptides were found in the hybrid but not in the hamster cell. Some, but not all, of these peptides segregated with markers for chromosome 21. Hamster peptides were also found which apparently were suppressed in the hybrid. Finally, one peptide was identified which was unique to the revertant cell. These findings may be of potential relevance to Down's syndrome.     

Crystallization of DNA fragments from water-salt solutions,containing 2-methylpentane-2,4-diol

Fragments of calf thymus DNA have been crystallized by precipitation from water-salt solutions, containing 2-methylpentane-2,4-diol (MPD). DNA crystals usually take the form either of spherulites up to 100 in diameter or of needles with the length up to 50 . No irreversible denaturation of DNA occurs during the crystallization process. X-ray diffraction from dense slurries of DNA crystals yields crystalline powder patterns.     

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE): advances and perspectives

The recent trend in science is to assay as many biological molecules as possible within a single experiment. This trend is evident in proteomics where the aim is to characterize thousands of proteins within cells, tissues, and organisms. While advances in mass spectrometry have been critical, developments made in two-dimensional PAGE (2D-PAGE) have also played a major role in enabling proteomics. In this review, we discuss and highlight the advances made in 2D-PAGE over the past 25 years that have made it a foundational tool in proteomic research.     

Accurate evaluation of the sizes of DNA fragments (from 30 to 4700 kb) in pulse field gel electrophoresis.

Construction of long-range genomic maps by pulse field gel electrophoresis requires optimum resolution of large DNA fragments. Using the transverse-alternating field electrophoresis system, we describe a method to accurately evaluate the sizes of fragments generated by rare-cutter digestions within the 30-4700-kb range. A protocol generating large (greater than 1000 kb) molecules by partial digestion is also reported.     

DNA binding and gel electrophoresis studies of a new silver(I) complex containing 2,9-dimethyl-1,10-phenanthroline ligands

The silver(I) complex, [Ag(2,9-dimethyl-1,10-phenanthroline)(2)](NO(3)) · H(2)O, has been synthesized and characterized by physicochemical and spectroscopic methods. The binding interactions of this complex with calf thymus DNA (CT-DNA) were investigated using emission, absorption, circular dichroism, viscosity measurements, and gel electrophoresis studies. The calculated binding constant, K(b), obtained from UV-vis absorption studies was 5.3 ± 0.2 × 10(4) M(-1). In fluorimetric studies, the enthalpy and entropy of the reaction between the complex and CT-DNA showed hydrophobic interaction. In addition, in the circular dichroism spectrum, silver(I) complex induces a B → A structural transition of CT-DNA. Gel electrophoresis studies demonstrated that this complex has ability to cleave the supercoiled plasmid DNA. All these results suggest that the complex interacts with CT-DNA via partial intercalative mode of binding.     

New complex of post-activated neocarzinostatin chromophore with DNA: bulge DNA binding from the minor groove

Neocarzinostatin (NCS-chrom), a natural enediyne antitumor antibiotic, undergoes either thiol-dependent or thiol-independent activation, resulting in distinctly different DNA cleavage patterns. Structures of two different post-activated NCS-chrom complexes with DNA have been reported, revealing strikingly different binding modes that can be directly related to the specificity of DNA chain cleavage caused by NCS-chrom. The third structure described herein is based on recent studies demonstrating that glutathione (GSH) activated NCS-chrom efficiently cleaves DNA at specific single-base sites in sequences containing a putative single-base bulge. In this structure, the GSH post-activated NCS-chrom (NCSi-glu) binds to a decamer DNA, d(GCCAGAGAGC), from the minor groove. This binding triggers a conformational switch in DNA from a loose duplex in the free form to a single-strand, tightly folded hairpin containing a bulge adenosine embedded between a three base pair stem. The naphthoate aromatic moiety of NCSi-glu intercalates into a GG step flanked by the bulge site, and its substituent groups, the 2-N-methylfucosamine carbohydrate ring and the tetrahydroindacene, form a complementary minor groove binding surface, mostly interacting with the GCC strand in the duplex stem of DNA. The bulge site is stabilized by the interactions involving NCSi-glu naphthoate and GSH tripeptide. The positioning of NCSi-glu is such that only single-chain cleavage via hydrogen abstraction at the 5'-position of the third base C (which is opposite to the putative bulge base) in GCC is possible, explaining the observed single-base cleavage specificity. The reported structure of the NCSi-glu-bulge DNA complex reveals a third binding mode of the antibiotic and represents a new family of minor groove bulge DNA recognition structures. We predict analogue structures of NCSi-R (R = glu or other substituent groups) may be versatile probes for detecting the existence of various structures of nucleic acids. The NMR structure of this complex, in combination with the previously reported NCSi-gb-bulge DNA complex, offers models for specific recognition of DNA bulges of various sizes through binding to either the minor or the major groove and for single-chain cleavage of bulge DNA sequences.     

Two-dimensional difference gel electrophoresis (DIGE) analysis of sera from visceral leishmaniasis patients

Introduction  

Visceral leishmaniasis is a parasitic infection caused by Lesihmania donovani complex and transmitted by the bite of the phlebotomine sand fly. It is an endemic disease in many developing countries with more than 90% of the cases occurring in Bangladesh, India, Nepal, Sudan, Ethiopia and Brazil. The disease is fatal if untreated. The disease is conventionally diagnosed by demonstrating the intracellular parasite in bone marrow or splenic aspirates. This study was carried out to discover differentially expressed proteins which could be potential biomarkers.     

Interaction of polypyridyl ruthenium (II) complex containing asymmetric ligand with DNA

A novel asymmetric bidentate ruthenium (II) complex, [Ru(bpy)(2)(PYNI)](2+) (bpy=2,2'-bipyridine, PYNI=2-(2'-pyridyl)naphthoimidazole), has been synthesized and characterized by elemental analysis, ES-MS (electrospray mass spectra) and (1)H NMR. The electrochemical behaviors of this complex were studied by cyclic voltammetry. DNA interaction studies suggest that [Ru(bpy)(2)(PYNI)](2+) binds to calf thymus DNA (CT-DNA) in an intercalative mode. Interestingly, this new Ru(II) complex has also been found to promote cleavage of plasmid pBR 322 DNA from the supercoiled form I to the open circular form II upon irradiation.     

Preferential hydrolysis of gap and bulge sites in DNA by Ce(IV)/EDTA complex

A new strategy for site-selective DNA hydrolysis, which takes advantage of the difference in reactivity between the phosphodiester linkages at the target site and the others, is presented. As the molecular scissors, homogeneous Ce(IV)/ethylenediamine-N,N,N′,N′-tetraacetate (EDTA) complex is used without being bound to any sequence-recognizing moiety. When a gap structure is formed at the target site by using two short oligonucleotides and the composite is treated with the Ce(IV)/EDTA complex at pH 7.0 and 37°C, the gap site in the substrate DNA is preferentially hydrolyzed over the double-stranded portion of the DNA. Site-selective DNA scission is also achieved by forming a bulge structure at the target site with the use of the appropriate oligonucleotide. These site-selective scissions are based on the following two factors: (i) the phosphodiester linkages in a single-stranded DNA are far more susceptible to the hydrolysis by the Ce(IV) complex than are the linkages in double-stranded DNA, and (ii) the phosphodiester linkages in the bulge sites are still more reactive than those in single-stranded DNA. In both cases, the addition of spermine significantly accelerates the scission.     

Two-dimensional display of lambda and Escherichia coli restriction fragments separated by Hg-Cs2SO4 centrifugation and gel electrophoresis

EcoRI restriction fragments derived from the DNA of bacteriophage lambda and Escherichia coli were fractionated by density gradient centrifugation of their mercury complexes in Cs2SO4 and subsequent electrophoresis on a horizontal agarose-gel slab. In this two-dimensional display, lambda fragments were resolved into six components and E coli fragments into more than 108 components. Bacterial chromosome regions contiguous to lambda prophage integrated at different sites were amplified by induction, and the EcoRI fragments were subjected to the two-dimensional analysis. As expected, the sets of amplified fragments were clearly different among the various lysogens. The approximate genome region affected by induction was estimated as one-tenth of the whole chromosome.     

Pulsed field gel electrophoresis techniques for separating 1- to 50-kilobase DNA fragments

Conventional agarose gel electrophoresis separates DNA using a static electric field. The maximum size limit for separation of DNA by this method is about 20 kilobase pairs (kb). A number of new electrophoretic techniques which employ periodic reorientation of electric fields permit separation of DNA well beyond this size limit. We sought to determine whether the use of very fast (millisecond) field switching could improve separation of DNA in the size range of 1 to 50 kb. Additionally, we have compared the resolution obtained with each of the different field switching regimens for DNA in this size range. Switching intervals of from 0.2 to 900 ms were used with unidirectional pulsing of a single electric field, with pulsed field gels, and with field inversion gel electrophoresis. Plotting the mobility of DNA as a function of size demonstrates that under the conditions used, each of these techniques offers comparable resolution. We also have examined the separation obtained when field inversion gels are run with forward and reverse fields of equal voltage and different durations, versus using fields of equal duration and different voltages. Field inversion which uses forward and reverse fields of different voltages yields resolution which is superior to the other methods examined.     

The irreversible binding of azacytosine-containing DNA fragments to bacterial DNA(cytosine-5)methyltransferases

DNA containing 5-azacytosine is an irreversible inhibitor of DNA(cytosine-5)methyltransferase. This paper describes the binding of DNA methyltransferase to 32P-labeled fragments of DNA containing 5-azacytosine. The complexes were identified by gel electrophoresis. The EcoRII methyltransferase specified by the R15 plasmid was purified from Escherichia coli B(R15). This enzyme methylates the second C in the sequence CCAGG and has a molecular mass of 60,000 Da. Specific binding of enzyme to DNA fragments could be detected if either excess unlabeled DNA or 0.8% sodium dodecyl sulfate was added to the reaction mixture prior to electrophoresis. Binding was dependent upon the presence of both the CCAGG sequence and azacytosine in the DNA fragment. S-Adenosylmethionine stimulated the formation of the complex. The complex was stable to 6 M urea but could be digested with pronase. These DNA fragments could be used to detect the presence of several different methyltransferases in crude extracts of E. coli. No DNA protein complexes could be detected in E. coli B extracts, a strain that contains no DNA(cytosine-5)methyltransferases. The chromosomally determined methylase with the same specificity as the purified EcoRII methylase could be detected in crude extracts of E. coli K12 strains. The MspI methylase cloned in E. coli HB101 could also be detected in crude extracts. These enzymes are the only proteins that bind azacytosine-containing DNA in crude extracts of E. coli.     

Interfacial thickness of liposomes containing poly(ethylene glycol)-cholesterol from electrophoresis.

The electrophoretic mobilities of liposomes incorporating a polyethylene glycol (PEG) headgroup coupled to cholesterol for PEG of average chain index 3.0, 13.2, and 22.3 have been determined as a function of PEG-cholesterol mole fraction between 5% and 40% and ionic strength between 2 and 200 mM. The liposome compositions were 40 mole % cholesterol plus PEG-cholesterol, 10 mole % 1,2-dipalmitoyl-sn-glyerco-3-phosphoglycerol, and 50 mole % egg phosphatidylcholine. The mobilities were fit to a model in which the PEG forms a surface layer of polymer subject to viscous drag arising from electroosmotic flow within this layer. The model provides estimates of the average layer thickness that are comparable to those determined from contemporary models of surface-attached polymer.     

Two-dimensional differential in gel electrophoresis (2D-DIGE) analysis of grape berry proteome during postharvest withering

The practice of postharvest withering is commonly used to correct quality traits and sugar concentration of high quality wines. To date, changes in the metabolome during the berry maturation process have been well documented; however, the biological events which occur at the protein level have yet to be fully investigated. To gain insight into the postharvest withering process, we studied the protein expression profiles of grape (Corvina variety) berry development focusing on withering utilizing a two-dimensional differential in gel electrophoresis (2D-DIGE) proteomics approach. Comparative analysis revealed changes in the abundance of numerous soluble proteins during the maturation and withering processes. On a total of 870 detected spots, 90 proteins were differentially expressed during berry ripening/withering and 72 were identified by MS/MS analysis. The majority of these proteins were related to stress and defense activity (30%), energy and primary metabolism (25%), cytoskeleton remodelling (7%), and secondary metabolism (5%). Moreover, this study demonstrates an active modulation of metabolic pathways throughout the slow dehydration process, including de novo protein synthesis in response to the stress condition and further evolution of physiological processes originated during ripening. These data represent an important insight into the withering process in terms of both Vitis germplasm characterization and knowledge which can assist quality improvement.     

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.