Biomedical subjects

N C Stellwagen

Publications and source records attributed to N C Stellwagen.

At least 19 recordsLinked to original sources

PubMed2001

Measuring the translational diffusion coefficients of small DNA molecules by capillary electrophoresis.

The apparent translational diffusion coefficients of four 20 base pair (bp) DNA oligonucleotides with different sequences have been measured by capillary electrophoresis, using the stopped migration method. The diffusion coefficients of the four oligomers were equal within experimental error, and averaged (120 +/- 10) x 10(-8) cm(2) s(-1) in 40 mM Tris-acetate-EDTA buffer at 25 degrees C. Since this value is nearly identical to the translational diffusion coefficient determined for a different 20-bp oligomer using other methods, the stopped migration method can accurately measure the diffusion coefficients of small DNA oligomers. The apparent diffusion coefficient of a 118-bp DNA restriction fragment was also measured by the stopped migration method. However, the observed value was approximately 25% larger than expected from other measurements, possibly because the diffusion coefficients of larger DNA molecules are somewhat dependent on the ionic strength of the solution.

DNA↗

PubMed2001

Preferential counterion binding to A-tract DNA oligomers.

The free solution mobility of four 20 bp DNA oligomers, with and without A-tracts, has been measured by capillary electrophoresis in Tris-acetate buffer, to test the hypothesis that site-specific binding of monovalent counterions can occur in the narrow minor groove of A-tract DNAs. Preferential counterion binding has been proposed to cause A-tract bending because of asymmetric charge neutralization and collapse of the helix backbone toward the minor groove. Preferential counterion binding in A-tract DNAs should be manifested by a decrease in the electrophoretic mobility observed in free solution, compared to that of non-A-tract DNAs of the same size. Of the four sequences studied here, the slowest absolute mobility, indicative of the greatest counterion binding, was observed for a 20 bp oligomer containing two runs of A3T3 in phase with the helix repeat. A 20-mer containing phased CACA sequences migrated with the fastest mobility; 20-mers containing phased A5 tracts or phased runs of T3A3 migrated with intermediate mobilities. Very similar mobility differences were observed when 1-20 mM NaCl was added to the buffer. The results suggest that preferential counterion binding occurs in A-tract DNAs, especially those containing the AnTn sequence motif.

Base Sequence↗

PubMed2001

Do orientation effects contribute to the molecular weight dependence of the free solution mobility of DNA?

The free solution mobility of DNA increases with increasing molecular weight and then levels off and becomes constant at molecular weights above approximately 400 bp (Stellwagen, N. C., Gelfi, C., Righetti, P. G., Biopolymers 1997,42, 687-703). To investigate whether the increase in mobility could be attributed to an increased orientation of the larger DNA molecules in the electric field, the free solution mobility of DNA was measured by capillary electrophoresis as a function of electric field strength. Mixtures containing 20-, 118- and 422-bp DNA molecules, and 20-, 422- and 2116-bp DNAs, were studied. If the larger DNA molecules in each mixture were oriented by the electric field, their mobilities should increase with electric field strength faster than the mobility of the 20-bp oligomer, which is too small to be oriented by the electric fields used in this study. Instead, the ratios of the mobilities of the 118-, 422- and 2116-bp fragments to the mobility of the 20-bp oligomer were independent of electric field strength. Hence, orientation effects are not important for DNA molecules up to 2 kbp in size, in electric fields up to 500 V/cm in amplitude. An explanation is suggested.

Base Sequence↗

PubMed2000

DNA and buffers: are there any noninteracting, neutral pH buffers?

The interaction of DNA with various neutral pH, amine-based buffers has been analyzed by free solution capillary electrophoresis, using a mixture of a plasmid-sized DNA molecule and a small DNA oligonucleotide as the reporter system. The two DNAs migrate as separate, nearly Gaussian-shaped peaks in 20-80 mM TAE (TAE, Tris-acetate-EDTA; Tris, tris[hydroxymethyl]aminomethane) buffer. The separation between the peaks gradually increases with increasing TAE buffer concentration because of differences in solvent friction between large and small DNA molecules. The two DNAs form complexes with the borate ions in TBE (Tris-borate-EDTA) buffer, with mobilities that depend on the DNA/borate ratio. In 45 mM TBE buffer, the two DNAs comigrate as a single sharp peak, with a mobility that is faster than either of the constituent DNAs in the same buffer. Hence, the mixed DNA-borate complex is stabilized by the binding of additional borate ions, possibly forming bridges between the different DNAs. The mixed DNA-borate complex is gradually dissociated into its component DNAs by increasing the TBE concentration, possibly because the borate binding sites become saturated at high buffer concentrations. Other neutral pH, amine-based buffers, such as Mops (3-[N-morpholino]propanesulfonic acid), Hepes (N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]), Bes (N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid), Tes (N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid), and tricine (N-tris[hydroxymethyl]methylglycine) also form complexes with DNA, giving distorted peaks in the electropherograms. The combined results indicate that borate buffers and most neutral pH, amine-based buffers interact with DNA.

Acetates↗

PubMed2000

Free solution mobility of DNA molecules containing variable numbers of cationic phosphoramidate internucleoside linkages.

The free solution electrophoretic mobility of an 118-base pair DNA fragment containing zero, three, six or nine cationic phosphoramidate internucleoside linkages has been measured by capillary electrophoresis. The electrophoretic mobility decreases with the increasing number of cationic phosphoramidate linkages, as expected because of the reduced negative charge on the DNA molecules. The decrease in mobility is approximately linear for DNA molecules containing three and six cationic phosphoramidate linkages, but begins to level off when nine cationic phosphoramidate linkages have been added. The mobility also varies somewhat depending on whether the modified phosphoramidate linkages are located at the 5'- or 3'-end of the DNA molecule.

Base Sequence↗

PubMed2000

DNA and buffers: the hidden danger of complex formation.

The free solution electrophoretic mobility of DNA differs significantly in different buffers, suggesting that DNA-buffer interactions are present in certain buffer systems. Here, capillary and gel electrophoresis data are combined to show that the Tris ions in Tris-acetate-EDTA (TAE) buffers are associated with the DNA helix to approximately the same extent as sodium ions. The borate ions in Tris-borate-EDTA (TBE) buffers interact with DNA to form highly charged DNA-borate complexes, which are stable both in free solution and in polyacrylamide gels. DNA-borate complexes are not observed in agarose gels, because of the competition of the agarose gel fibers for the borate residues. The resulting agarose-borate complexes increase the negative charge of the agarose gel fibers, leading to an increased electroendosmotic flow of the solvent in agarose-TBE gels. The combined results indicate that the buffers in which DNA is studied cannot automatically be assumed to be innocuous.

Acetates↗

PubMed2000

Conformational isomers of curved DNA molecules can be observed by polyacrylamide gel electrophoresis.

Dimers, trimers and higher multimers of two 147-base pair restriction fragments called 12 A and 12B, obtained from the MspI digest of plasmid pBR322, migrate as sharp bands in agarose and dilute polyacrylamide gels, indicating that they are homogeneous in molecular weight. However, the electrophoretic bands corresponding to multimers of the curved fragment 12A are split into sharp sub-bands in more concentrated polyacrylamide gels. The relative intensities and spacing of the sub-bands depend on the number of monomers in the multimer, the pH of the buffer, and the presence or absence of divalent cations in the solution. Since band splitting is not observed for the normal 12B multimers under any gel-running conditions, the sub-bands observed for multimers of the curved fragment 12A must be attributed to conformational isomers which are in slow exchange on the electrophoretic time scale. Band splitting is also observed for multimers of a curved DNA fragment containing the kinetoplast bending locus and for plasmid pUC19 linearized by digestion with certain restriction enzymes. Plasmid pUC19 contains two nearly equidistant regions of intrinsic curvature (Strutz, K., Stellwagen, N. C., Electrophoresis 1996, 17, 989-995). Hence, DNA molecules containing two or more regions of curvature exist as discrete subpopulations of conformational isomers which can be observed as separate bands migrating in polyacrylamide gels.

DNA↗

PubMed1999

DNA-histidine complex formation in isoelectric histidine buffers.

The free solution electrophoretic mobility of two DNA molecules of different molecular masses, 18 base pairs and 2686 base pairs, has been measured in isoelectric histidine buffers with and without added low-molecular-mass electrolytes. Extensive DNA-histidine complex formation is observed in isoelectric histidine buffer, as evidenced by distortion and splitting of the peaks in the electropherograms. Peak distortion and splitting can be decreased or eliminated by adding low-molecular-mass neutral salts to the solution, suggesting that the DNA-histidine complexes are stabilized by electrostatic interactions. The ability of various neutral salts to disrupt the DNA-histidine complexes depends on the molecular mass of the DNA and the concentration and type of added salt.

Anions↗

PubMed1998

Do DNA gel electrophoretic mobilities extrapolate to the free-solution mobility of DNA at zero gel concentration?

The electrophoresis of small DNA fragments has been measured in dilute agarose and polyacrylamide gels cast and run in Tris-acetate-EDTA (TAE) and Tris-borate-EDTA (TBE) buffers. Ferguson plots were constructed to extrapolate the mobilities to zero gel concentration and estimate the free solution mobility of DNA. In polyacrylamide gels, in both TAE and TBE buffers, the extrapolated mobilities at zero gel concentration increased gradually with decreasing DNA molecular weight, went through a maximum at approximately 60 bp, and then decreased again. The increase in the extrapolated mobilities with decreasing molecular weight observed for DNA fragments > or = 60 bp can be attributed to transient interactions between the migrating DNA molecules and the polyacrylamide gel fibers. If such interactions are eliminated by extrapolating the mobilities to both zero gel concentration and zero DNA molecular weight, the apparent free solution mobility of DNA is found to be 3.1 x 10(-4) cm2 V(-1) s(-1) in TAE buffer and 4.2 x 10(-4) cm2 V(-1) s(-1) in TBE buffer at 20 degrees C, reasonably close to the actual free solution mobilities measured in the same two buffers by capillary electrophoresis (N. C. Stellwagen et al., Biopolymers 1997, 42, 687-703). The significantly larger electrophoretic mobility observed in TBE buffer is most likely due to the formation of nonspecific, highly charged deoxyribose-borate complexes in this buffer medium. For DNA molecules < or = 60 bp in size, the decrease in the extrapolated mobilities with decreasing molecular weight parallels the decrease in their free solution mobilities observed by capillary electrophoresis. In agarose gels, the extrapolated mobilities of small DNA molecules at zero gel concentration appear to be independent of molecular weight. The apparent free solution mobilities are found to be (3.0 +/- 0.1) x 10(-4) cm2 V(-1) s(-1) in TAE buffer and (3.2 +/- 0.1) x 10(-4) cm2 V(-1) s(-1) in TBE buffer. The very similar mobilities observed in the two buffer media suggest that the borate ions in TBE buffer primarily form complexes with the galactose residues in the agarose gel fibers, rather than with the migrating DNA molecules, because of mass action effects. The formation of borate-agarose complexes, increasing the net negative charge of the agarose gel fibers, appears to be responsible for the markedly increased electroendosmotic flow observed in agarose gels cast and run in TBE buffer (N. C. Stellwagen, Electrophoresis 1992, 13, 601-603).

Acetates↗

PubMed1998

Apparent pore size of polyacrylamide gels: comparison of gels cast and run in Tris-acetate-EDTA and Tris-borate-EDTA buffers.

The electrophoretic mobilities of DNA molecules in three different molecular weight ladders were measured in polyacrylamide gels containing different acrylamide concentrations (%T) and cross-linker ratios (%C), cast and run in Trisacetate-EDTA (TAE) buffer. The apparent pore radius of each gel was estimated from Ferguson plots of the relative mobilities of each of the DNA molecules, using the mobility of the monomer fragment in each molecular weight ladder as the reference mobility. The effective size of each of the DNA molecules was estimated from its radius of gyration. The apparent gel pore radii calculated in this manner ranged from 21 nm in gels containing 10.5%T, 5%C to 200 nm in gels with 4.6%T, 2%C, similar to the values observed for polyacrylamide gels cast and run in Tris-borate-EDTA (TBE) buffer (Holmes and Stellwagen, Electrophoresis 1991, 12, 612-619). Hence, the effective pore size of polyacrylamide gels is essentially independent of whether the gels are cast and run in TAE or TBE buffer.

Acetates↗

PubMed1998

Capillary zone electrophoresis of ds-DNA in isoelectric buffers: effect of adding of competing, nonamphoteric ions.

When separating ds-DNA in isoelectric His buffer (pH=pI=7.6), in the 50-250 mM concentration range, some unique phenomena were observed: improved resolution for smaller DNA fragments, up to ca. 150 bp, and a rapid deterioration of resolution above this critical length (which corresponds to the persistence length). Such phenomenon depended also on voltage and concentration of sieving liquid polymer. Direct binding of His to the DNA helix was hypothesized, with resultant stiffening and an increment of diameter of the DNA fragments, thus inducing an early onset of reptation at the applied voltage in the 100-300 V/cm range. In order to prove this hypothesis, "competing ions" (notably NaCl and KBr) were added to the His background electrolyte: a partial reversal of the His effect was already apparent at low concentrations of such ions (10 mM) and was complete at higher concentrations (30 and 50 mM). By molecular modeling, it was found that His could be docking on the negatively charged oxygen (bound to the phosphate) by offering both charged (primary and tertiary amino) groups to simultaneous binding, thus forming a salt and neutralizing the negative charge borne by the oxygen. The following characteristic bond distances were found: 0.34 nm between the N (imidazolic) and O; 0.32 nm between the primary N and O; 0.36 nm between the two nitrogens engaged in salt formation with the oxygen. In addition, for complexation to occur, the distance between the noncharged nitrogen in the imidazole ring and the nearest phosphate oxygen (engaged in the phosphodiester bridge) should be 0.44 nm. Under these conditions, the two rings present (a six-membered, ideal one, salt-linked with the oxygen and rather highly elongated, and the imidazole) will not be precisely coplanar, since the primary and tertiary nitrogens will be one slightly above and one slightly below the plane of the drawing. Upon extensive binding, occupying every available phosphate site, pi-pi interactions could occur among the stacks of bound His residues, thus further stabilizing the complex.

Buffers↗

PubMed1997

The free solution mobility of DNA.

The free solution mobility of DNA has been measured by capillary electrophoresis in the two buffers most commonly used for DNA gel electrophoresis, Tris-borate-EDTA (TBE) and Tris-acetate-EDTA (TAE). The capillaries were coated with polymers of either of two novel acrylamide monomers, N-acryloylaminoethoxyethanol or N-acryloylaminopropanol, both of which are stable at basic pH and effectively eliminate the electroendosmotic mobility due to the capillary walls. The free solution mobility of DNA in TAE buffer was found to be (3.75 +/- 0.04) x 10(-4) cm2 V-1 s-1 at 25 degrees C, independent of DNA concentration, sample size, electric field strength, and capillary coating, and in good agreement with other values in the literature. The free solution mobility was independent of DNA molecular weight from approximately 400 base pairs to 48.5 kilobase pairs, but decreased monotonically with decreasing molecular weight for smaller fragments. Surprisingly, the free solution mobility of DNA in TBE buffer was found to be (4.5 +/- 0.1) x 10(-4) cm2 V-1 s-1, about 20% larger than observed in TAE buffer, presumably because of the formation of nonspecific borate-deoxyribose complexes.

DNA↗

PubMed1997

DNA mobility anomalies are determined primarily by polyacrylamide gel concentration, not gel pore size.

The dependence of DNA mobility anomalies on gel pore size has been studied in polyacrylamide gels with a wide variety of compositions, using molecular weight ladders containing multiple copies of normal (12B) and anomalously slowly migrating (12A) 147-base pair restriction fragments from plasmid pBR322 as the migrating probe molecules. If the gel pore size is increased by decreasing the total acrylamide concentration (%T) at constant cross-linker ratio (%C), the usual method of increasing gel pore size, the mobility anomalies decrease with increasing gel pore radius as though the 12A multimers were retarded by a sieving mechanism. However, the decrease in the mobility anomalies is independent of whether the apparent gel pore radius is larger or smaller than the DNA radius of gyration, suggesting that gel pore size is not the controlling variable. If the acrylamide concentration is held constant and the gel pore size is increased by decreasing %C at constant %T, the mobility anomalies of the largest 12A multimers (6 mers and higher) decrease with increasing gel pore radius, because of sieving effects, until the effective gel pore radius becomes approximately equal to the DNA radius of gyration, after which the mobility anomalies level off and become independent of gel pore size. The mobility anomalies exhibited by 5-mers and smaller multimers of fragment 12A are independent of gel pore radius in all gels with constant %T. Similar results are observed with a molecular weight ladder containing phased A-tracts from the kinetoplast bending locus. Since the anomalous electrophoretic mobilities depend primarily on the total acrylamide concentration in the gel, and not on the apparent gel pore radius, increases in the magnitude of the mobility anomalies with increasing gel concentration (and decreasing gel pore radius) cannot be taken as evidence for DNA curvature.

Acrylic Resins↗

PubMed1996

Electric birefringence of kilobase-sized DNA molecules.

Transient electric birefringence has been used to characterize the rotational diffusion of linear, circularly permuted pBR322 and SV40 DNA molecules. The birefringence relaxation times vary with the site of linearization, suggesting that the circularly permuted DNAs have different conformations in solution. The longest relaxation times are observed for DNA sequence isomers linearized at the major bend centers identified by gel electrophoresis. SV40 sequence isomers linearized at other locations have faster, but approximately equal, terminal relaxation times, suggesting that their free solution conformations are relatively independent of the location of the bend center within the sequence. By contrast, the terminal relaxation times of the various pBR322 sequence isomers vary approximately in accord with their electrophoretic mobilities in large-pore polyacrylamide gels, suggesting that the different mobilities may reflect real conformational differences between the sequence isomers.

Birefringence↗

PubMed1996

Intrinsic curvature of plasmid DNAs analyzed by polyacrylamide gel electrophoresis.

The electrophoretic mobility of two small DNA plasmids, pUC19 and Litmus 28, linerarized by digestion with a variety of single-cut restriction enzymes, has been studied. The permuted sequence isomers migrate with identical mobilities in agarose gels, as expected, but exhibit different mobilities in large-pore polyacrylamide gels, suggesting that the parent plasmids contain sequence-specific sites of curvature and/or anisotropic flexibility. Both plasmids contain apparent bend centers near their origin(s) of replication; pUC19 also has a major apparent bend center near the promoter of the ampicillin resistance gene. These apparent bend centers are observed under a variety of experimental conditions, suggesting that they correspond to sites of stable curvature in the parent plasmids. Both plasmids also contain minor bend centers that are observed under a sub-set of electrophoretic conditions and disappear when divalent cations are added to the solution, suggesting that these apparent bend centers may correspond to localized regions of variable flexibility.

DNA, Circular↗

PubMed1995

Use of polyacrylamide gel electrophoresis to detect structural variations in kilobase-sized DNAs.

The electrophoresis of linear, kilobase-sized DNA molecules with permuted sequences has been studied in polyacrylamide and agarose gels. Plasmid pBR322, bacteriophage phi X174, and the SV40 minichromosome were each digested with a series of single-cut restriction enzymes. The linearized, permuted isomers of all three DNAs exhibit different mobilities in large-pore polyacrylamide gels, suggesting that all three DNAs contain sites of anisotropic, sequence-dependent curvature. Various experimental parameters such as acrylamide concentration, crosslinker ratio and buffer composition affect the magnitude of the observed differential mobilities. Band sharpness appears to be optimal in polyacrylamide gels containing 6.9-8.1%T and 0.5-1%C. Only small mobility differences are observed for the linearized, permuted sequence isomers in agarose gels.

Animals↗

PubMed1995

Effect of spike pulses on the orientation of the agarose gel matrix.

The orientation of the agarose gel matrix in two-part, "stair-step" electric fields has been studied by transient electric birefringence. Stair-step electric fields are those in which a pulse of a given amplitude is immediately followed by a short, higher voltage "spike" pulse of the same polarity. A single stair-step pulse orients the agarose gel matrix as though the two portions of the pulse were individually applied to the gel. However, a series of consecutive stair-step pulses causes an anomalous increase in the amplitude of the birefringence, suggesting that increased numbers of agarose fiber bundles are orienting in the electric field. Spike pulses > or = 10 V/cm appear to cause junction zone breakdown, freeing large numbers of agarose fiber bundles and microgel domains from the constraints of the gel matrix. The implications of these results for pulsed field gel electrophoresis are discussed.

Birefringence↗

PubMed1994

Transient electric birefringence of agarose gels. I. Unidirectional electric fields.

The orientation of agarose gels in pulsed electric fields has been studied by the technique of transient electric birefringence. The unidirectional electric fields ranged from 2 to 20 V/cm in amplitude and 1 to 100 s in duration, values within the range typically used for pulsed field gel electrophoresis (PFGE). Agarose gels varying in concentration from 0.3 to 2.0% agarose were studied. The sign of the birefringence varied randomly from one gel to another, as described previously [J. Stellwagen & N.C. Stellwagen (1989), Nucleic Acids Research, Vol. 17, 1537-1548]. The sign and amplitude of the birefringence also varied randomly at different locations within each gel, indicating that agarose gels contain multiple subdomains that orient independently in the electric field. Three or four relaxation times of alternating sign were observed during the decay of the birefringence. The various relaxation times, which range from 1 to approximately 120 s, can be attributed to hierarchies of aggregates that orient in different directions in the applied electric field. The orienting domains range up to approximately 22 microns in size, depending on the pulsing conditions. The absolute amplitude of the birefringence of the agarose gels increased approximately as the square of the electric field strength. The measured Kerr constants are approximately 5 orders of magnitude larger than those observed when short, high-voltage pulses are applied to agarose gels. The increase in the Kerr constants in the low-voltage regime parallels the increase in the relaxation times in low-voltage electric fields. Birefringence saturation curves in both the low- and high-voltage regimes can be fitted by theoretical curves for permanent dipole orientation. The apparent permanent dipole moment increases approximately as the 1.6 power of fiber length, consistent with the presence of overlapping agarose helices in the large fiber bundles orienting in low-voltage electric fields. The optical factor is approximately independent of fiber length. Therefore, the marked increase in the Kerr constants observed in the low-voltage regime is due to the large increase in the electrical orientation factor, which is due in turn to the increased length of the fiber bundles and domains orienting in low-voltage electric fields. Since the size of the fiber bundles and domains approximates the size of the DNA molecules being separated by PFGE, the orientation of the agarose matrix in the applied electric field may facilitate the migration of large DNA molecules during PFGE.

Birefringence↗