DNA Chain Length Research Articles

Conformational and thermodynamic properties of supercoiled DNA.

We used Monte Carlo simulations to investigate the conformational and thermodynamic properties of DNA molecules with physiological levels of supercoiling. Three parameters determine the properties of DNA in this model: Kuhn statistical length, torsional rigidity and effective double-helix diameter. The chains in the simulation resemble strongly those observed by electron microscopy and have the conformation of an interwound superhelix whose axis is often branched. We compared the geometry of simulated chains with that determined experimentally by electron microscopy and by topological methods. We found a very close agreement between the Monte Carlo and experimental values for writhe, superhelix axis length and the number of superhelical turns. The computed number of superhelix branches was found to be dependent on superhelix density, DNA chain length and double-helix diameter. We investigated the thermodynamics of supercoiling and found that at low superhelix density the entropic contribution to superhelix free energy is negligible, whereas at high superhelix density, the entropic and enthalpic contributions are nearly equal. We calculated the effect of supercoiling on the spatial distribution of DNA segments. The probability that a pair of DNA sites separated along the chain contour by at least 50 nm are juxtaposed is about two orders of magnitude greater in supercoiled DNA than in relaxed DNA. This increase in the effective local concentration of DNA is not strongly dependent on the contour separation between the sites. We discuss the implications of this enhancement of site juxtaposition by supercoiling in the context of protein-DNA interactions involving multiple DNA-binding sites.

Nature of protamine-DNA complexes: A special type of ligand binding co-operativity

The mode of protamine binding to DNA double helices has been analyzed for the example of clupein Z from herring and DNA samples from bacteriophages λ and PM2 by measurements of light-scattering intensities, ultracentrifugation and kinetics. The light-scattering intensity of DNA increases co-operatively at a threshold clupein concentration suggesting co-operative binding of clupein to double helices. These data are first analyzed in terms of a model with a transition at a threshold degree of binding. The parameters resulting from this analysis appear to be reasonable, but are shown to be in contrast with data on the absolute degree of clupein binding to DNA obtained by centrifugation experiments. An analysis of the kinetics associated with clupein binding to DNA by measurements of the time-dependence of light-scattering intensities in the time range of seconds demonstrates directly that clupein-induced intermolecular interactions of DNA molecules are essential. The rate constants of DNA association increase co-operatively at threshold clupein concentrations, which correspond to those observed in the equilibrium titrations. Above the threshold, the rate constants arrive at a level that is almost constant, but shows some decrease with increasing clupein concentrations. These results are described by a model with a monomer and a dimer state of DNA, which bind ligands with different affinities according to an excluded-site binding scheme. When the ligand binding constant is larger for the dimer than for the monomer state, as should be expected, binding of ligands drives the DNA from the monomer to the dimer state, even if the dimerization equilibrium in the absence of ligands is far in favor of the monomer. The transition from the monomer to the dimer state proves to be strongly co-operative. When the ligand concentration is increased to higher values, the dimers may be converted back to monomers due to an increased extent of ligand binding to the monomer state. The model is consistent with the available experimental data. The analysis of the data by the model indicates the existence of a reaction unit much below the DNA chain length, corresponding to about 80 nucleotide residues. The present model describes ligand driven intermolecular association; an analogous model is applicable to ligand driven intramolecular association. In summary, the co-operativity of clupein binding to DNA double helices is not due to nearest neighbor interactions, but results from thermodynamic coupling of clupein binding with clupein-induced DNA association.

Synthesis of DNA by DNA polymerase epsilon in vitro.

The isolation of DNA polymerase (Pol) epsilon from extracts of HeLa cells is described. The final fractions contained two major subunits of 210 and 50 kDa which cosedimented with Pol epsilon activity, similar to those described previously (Syvaoja, J., and Linn, S. (1989) J. Biol. Chem. 264, 2489-2497). The properties of the human Pol epsilon and the yeast Pol epsilon were compared. Both enzymes elongated singly primed single-stranded circular DNA templates. Yeast Pol epsilon required the presence of a DNA binding protein (SSB) whereas human Pol epsilon required the addition of SSB, Activator 1 and proliferating cell nuclear antigen (PCNA) for maximal activity. Both enzymes were totally unable to elongate primed DNA templates in the presence of salt; however, activity could be restored by the addition of Activator 1 and PCNA. Like Pol delta, Pol epsilon formed complexes with SSB-coated primed DNA templates in the presence of Activator 1 and PCNA which could be isolated by filtration through Bio-Gel A-5m columns. Unlike Pol delta, Pol epsilon bound to SSB-coated primed DNA in the absence of the auxiliary factors. In the presence of salt, Pol epsilon complexes were less stable than they were in the absence of salt. In the in vitro simian virus 40 (SV40) T antigen-dependent synthesis of DNA containing the SV40 origin of replication, yeast Pol epsilon but not human Pol epsilon could substitute for yeast or human Pol delta in the generation of long DNA products. However, human Pol epsilon did increase slightly the length of DNA chains formed by the DNA polymerase alpha-primase complex in SV40 DNA synthesis. The bearing of this observation on the requirement for a PCNA-dependent DNA polymerase in the synthesis and maturation of Okazaki fragments is discussed. However, no unique role for human Pol epsilon in the in vitro SV40 DNA replication system was detected.

Detection of conformational and net charge differences in DNA‐protein complexes by quantitative electrophoresis on polyacrylamitie‐agarose copolymer gels

The Galactosidase repressor (GalR) of Escherichia coli modulates the expression of the gal operon by binding to two DNA operators, OE and O1. The OE and O1 elements are 16 bp pallindromic DNA sequences, differing in four of the base pairs. OE and O1 DNA fragments, both free and complexed with repressor, were analyzed by "quantitative gel electrophoresis". By the criteria of that method, applied to the linear Ferguson plots of both DNA fragments and the linear ranges of those of the DNA-GalR complexes, it was shown that the apparent size of DNA increases upon repressor binding. Moreover, this size increase is greater for the complex with the O1 operator than for the complex with the OE operator in the case that GalR is located in the center of a 155 bp DNA fragment. This is not the case when GalR is located in a peripheral position. By contrast with their size differences, the centrally located GalR-O1 and GalR-OE complexes appear to possess indistinguishable net surface charge densities as judged from the intercepts with the mobility axis. The larger size of the complex with centrally located O1 fragment, as compared with that bearing the OE fragment, is interpreted as being due to bending of the DNA-protein complex, since an authentically bent fragment of a plasmid with bent upstream activator sequence also exhibits a larger slope of the Ferguson plot, and thus the larger size, than predicted on the basis of its DNA chain length (bp).(ABSTRACT TRUNCATED AT 250 WORDS)

Single-stranded DNA-dependent ATPase from HeLa cells that stimulates DNA polymerase .alpha.-primase activity: purification and characterization of the ATPase

A single-stranded DNA-dependent ATPase that cofractionates during the early stages of purification of a multiprotein DNA polymerase alpha complex from HeLa cells has been purified to homogeneity. The ATPase is part of a 16S multienzyme DNA polymerase alpha complex that is fully active in SV40 DNA replication in vitro. The ATPase hydrolyzes ATP to ADP in a reaction that is completely dependent on the presence of DNA. DNA in single-stranded form is strongly preferred as a cofactor, and polydeoxynucleotides with adenine or thymidine residues are highly effective. Glycerol gradient sedimentation showed that the purified ATPase sedimented at an s20,w of 7 S, and polyacrylamide gel electrophoresis under denaturing conditions reveals two polypeptides with relative molecular weights of 83,000 and 68,000. Both of these polypeptides have purine nucleotide binding sites as revealed by photoaffinity cross-linking experiments. ATP binds to the two subunits more efficiently than GTP, and CTP or UTP does not cross-link with the two polypeptides. DNA synthesis catalyzed by purified HeLa cell DNA polymerase alpha-primase is stimulated in the presence of ATPase and ATP at an optimum concentration of 2 mM. Analysis of the DNA product by gel electrophoresis indicates that with poly(dT) but not phage M13 DNA as template the ATPase overcomes a lag and decreases the length of nascent DNA chains synthesized by the DNA polymerase alpha-primase complex.

Theory of resonant microwave absorption of DNA in aqueous solution

The Ostrovskii-Sutin equation of the longitudinal displacement of an elastic rod has been derived for a DNA chain by taking its structure into account. The perturbation solution of this equation shows that the resonant microwave absorption by DNA in solution is possible. In addition to the primary resonance at ωn, our theory predicts also a subharmonic resonance at ωn/2. The strength of the latter is proportional to l-3, where l is the length of DNA chain.

An unusual electrooptical effect observed for DNA fragments and its apparent relation to a permanent electric moment associated with bent DNA.

Dichroism decay curves of DNA fragments with chain lengths in the range of 179-256 bp show an amplitude inversion suggesting the existence of a positive dichroism component, when these fragments are dissolved at monovalent salt concentrations above approx. 5 mM and are exposed to field pulses with amplitudes and/or lengths above critical values. At the critical values, the unusual dichroism is reflected by an apparent acceleration of the decay curves, which can be fitted by single exponentials with time constants much below the values expected from the DNA contour lengths. The critical pulse amplitudes and lengths decrease with increasing DNA chain length and increasing salt concentration. The experimental data are consistent with results obtained by hydrodynamic and electric model calculations on smoothly bent DNA double helices. The DNA is represented by a string of overlapping beads, which is used to calculate the rotational diffusion tensor and the center of diffusion. The distribution of phosphate charges is asymmetric with respect to this center and thus gives rise to a substantial permanent dipole moment. The magnitude of this dipole moment is calculated as a function of DNA curvature and is used together with experimental values of polarizabilities for simulations of dichroism decay curves. The curves simulated for bent DNA show the same phenomenon as observed experimentally. The ionic strength dependence of the unusual dichroism is explained by an independently observed strong decrease of the polarizability with increasing salt concentration. The field strength dependence is probably due to field-induced bending of double helices driven by the change of the dipole moment. Although our calculations are on rigid models of DNA and thus any flexibility of the double helix has not been considered, we conclude that the essential part of our experimental results can be explained by our model.

Origin and degradation of the RNA primers at the 5′ termini of nascent DNA chains in Bacillus subtilis

We had earlier characterized the nascent DNA synthesized in permeable cells of Bacillus subtilis in the presence of 5-mercurideoxycytidine triphosphate and 2′,3′-dideoxyATP as being substituted at its 5′ end with a ribonucleotide moiety of the sequence pApG(pC) 1–2 DNA. In this paper, we examine the origin and turnover of the DNA-linked ribonucleotide and its relationship to DNA replication. At least 50% of the RNA-linked nascent DNA chains served as guanylate acceptors when incubated with GTP and the eukaryotic capping enzyme, indicating the presence of 5′-terminal di- or triphosphate groups and suggesting that the RNA moiety is synthesized de novo and is not a degradation product. In nascent DNA produced without limitation of chain growth by dideoxyATP, the degree of terminal ribonucleotide substitution was reduced by 50%, consistent with a linkage between RNA primer removal and DNA chain growth. Such a relationship was demonstrated directly by examining the RNA primer content of nascent DNA synthesized in the absence of dideoxyATP as a function of DNA chain length. As the DNA size increased from 40 to 200 nucleotide residues, the extent of RNA substitution declined from 80% to nearly 0%. Endgroup analysis showed that the loss of RNA was accompanied by a gradual shift from predominantly adenylate residues to 5′-terminal guanylate, consistent with a stepwise removal of ribonucleotides from the 5′ end. Evidence that the nascent mercurated DNA synthesized under our experimental conditions was indeed a replicative intermediate came from the study of the time course of DNA chain growth and pulse-chase experiments. In the presence of the DNA ligase inhibitor NMN, mercurated DNA accumulated in two size classes with average length of approximately 750 and 8000 nucleotide residues, presumably representing the mature size of intermediates in discontinuous DNA synthesis. Comparison with the DNA size range at which the loss of the 5′-terminal RNA moiety occurred (40 to 200 residues) indicated that the processing of RNA primers occurred at an early stage during DNA chain elongation, and that moderate size intermediates in discontinuous DNA replication (200 nucleotides) have already lost their RNA primers.

The role of palindromic and non-palindromic sequences in arresting DNA synthesis in vitro and in Vivo

The nature of specific DNA sequences that arrest synthesis by mammalian DNA polymerase α in vitro was analyzed using circular, single-stranded M13 or φX174 virion DNA templates annealed to a unique, terminally labeled, DNA primer. This method rigorously defined both the starting nucleotide position and the direction of synthesis, as well as making the amount of radioactivity proportional to the number rather than the length of nascent DNA chains. The precise nucleotide locations of arrest sites were determined over templates with complementary sequences by cloning unique DNA restriction fragments into M13 DNA and isolating virions containing either the Watson or Crick strand. Results were correlated with the locations of palindromic (self-complementary) sequences, repeated sequences, and repeated sequences with mirror-image orientation. Two classes of DNA synthesis arrest sites were identified, distinct in structure but equivalent in activity. Class I sites consisted of palindromic sequences that formed a stable hairpin structure in solution and arrested DNA polymerase on both complementary templates. The polymerase stopped precisely at the base of the duplex DNA stem, regardless of the direction from which the enzyme approached. Class II sites consisted of non-palindromic sequences that could not be explained by either secondary structure or sequence symmetry elements, and whose complementary sequence was not an arrest site. Size limits, orientation and some sequence specificity for arrest sites were suggested by the data. Arrest sites were also observed in vivo by mapping the locations of 3′-end-labeled nascent simian virus 40 DNA strands throughout the genome. Arrest sites closest to the region where termination of replication occurs were most pronounced, and the locations of 80% of the most prominent sites appeared to be recognized by α-polymerase on the same template in vitro. However, class I sites were not identified in vivo, suggesting that palindromic sequences do not form hairpin structures at replication forks.

Interactions of nucleic acid double helices induced by electric field pulses.

Electric field pulses induce a substantial increase of the light scattering intensity of double-helical DNA. The relative change of light scattering and also the reciprocal relaxation time constants under electric field pulses increase with increasing nucleotide concentration. These observations, together with a large difference between dichroism orientation time constants and light scattering time constants under electric field pulses, demonstrate that the main part of the light scattering effect is due not to field-induced orientation but to interactions between DNA helices. From the concentration dependence of the light scattering time constants we obtain, according to an isodesmic reaction model, association rate constants in the range 3 X 10(10) M-1 helices s-1 for DNA with approx. 300 base-pairs. These values are at the limit of a diffusion-controlled DNA association and do not show any dependence upon the field strength. The dissociation rate constants kd decrease strongly with increasing field strength E and thus demonstrate that the interactions between the helices are induced by the electric field. This conclusion is consistent with independent measurements which do not reveal any DNA association at zero field strength. The observed linear relation between log(kd) and E2 suggests a field-induced reaction driven by dipole changes. According to this interpretation the change of dipole moment should be in the range of approx. 1400 debye. The dissociation rates for DNA helices with approx. 300 to approx. 800 base-pairs strongly increase with increasing salt concentration (measured in the range 1-5 mM ionic strength), whereas the association rate constants remain virtually unchanged. Measurements of the linear dichroism in the same range of DNA chain length demonstrate that for long field pulses of e.g., 40 microseconds, the amplitude approaches a maximum value and then decreases. The dichroism relaxation curves observed after long field pulses exhibit a component with a positive dichroism and an increased decay time. These observations suggest the formation of a DNA aggregate with an unusual arrangement of the bases.

Two DNA methyltransferases from murine erythroleukemia cells: purification, sequence specificity, and mode of interaction with DNA.

Dye-ligand chromatography on Cibacron blue F3GA-agarose has been used to resolve two species of DNA (cytosine-5-)-methyltransferase from nuclear extracts of uninduced Friend murine erythroleukemia cells. Each species has been highly purified; the activities in the first and second peaks were associated with polypeptides of Mr 150,000 and 175,000, respectively. Analysis of substrate specificity with synthetic DNAs and restriction fragments of phi X174 replicative form DNA and pBR322 DNA showed that neither enzyme had dependence on the sequence context of CpG dinucleotides; poly(dG-dC) had the greatest methyl-accepting activity of any unmethylated DNA substrate tested. De novo methylation by both enzymes was inefficient relative to methylation of hemimethylated sites. Methyl-accepting activity was strongly dependent on DNA chain length. This observation suggests that binding to DNA, followed by one-dimensional diffusion of enzyme along the DNA molecule, is important in the mechanism by which DNA methyltransferase locates its recognition sites.

Three organizations of human DNA.

Human DNA was denatured, annealed to low C0t, and fractionated by centrifugation to equilibrium on gradients of NaI. Three well-defined zones resulted. The topmost, least dense zone contained satellite DNA. The bottom zone contained molecules bearing infrequent sequences and short repeated DNA. The middle zone contained molecules bearing infrequent sequences, short repeated DNA, and long repeated DNA. The NaI pattern was independent of single-strand DNA chain length from 5 to 30 kilobases. Structural genes were found in the organization bearing long repeats.

Rotational diffusion of Escherichia coli RNA polymerase free and bound to deoxyribonucleic acid in nonspecific complexes.

We have studied the rotational diffusion of Escherichia coli RNA polymerase free in solution and bound nonspecifically to DNA fragments. The rotational motion was measured by the decay in anisotropy of the triplet-triplet absorption by using as probes either the liganded enzyme inhibitor Rose Bengal or eosin 5'-isothiocyanate conjugated to the protein. The time resolution extended from 10 ns to 1 ms. Free RNA polymerase (holoenzyme) at high salt concentration (1 M NaCl) is monomeric and diffuses at 5 degrees C with a rotational correlation time of 0.66 microseconds, corresponding to an equivalent hydrodynamic sphere with a radius of 7.4 nm. These values and the known molecular weight are most compatible with a nonspherical shape, e.g., an oblate ellipsoid with an axial ratio of about 3. In 0.1 M NaCl, the holoenzyme is dimeric and has a rotational correlation time of 2 microseconds. The decay of anisotropy is at least biexponential upon binding RNA polymerase to calf thymus DNA or to poly[d(A-T)]. The fast component with half of the amplitude has decay kinetics comparable to those seen with the free monomeric enzyme. The slow component has a rotational correlation time of about 14 microseconds and is independent of DNA chain length in the range greater than 180 base pairs. Both rotational correlation times decrease with temperature, and the relative amplitudes change such that the faster component dominates at higher temperature. The rotational relaxation of the enzyme-DNA complexes is discussed in terms of alternative models involving rigid rod-sphere diffusion, conformational changes in the enzyme and/or DNA, sliding motions of the protein along the DNA, and torsional-bending motions of DNA envisioned as a deformable rod.

Analysis of the 5′-termini of nascent DNA chains synthesized in permeable cells of Bacillus subtilis

The nascent DNA synthesized by permeable cells of Bacillus subtilis in the presence of 5′-mercurideoxycytidine triphosphate and 2′,3′-dideoxyATP has been isolated and characterized. The newly synthesized DNA was isolated free from other cellular nucleic acids by affinity chromatography on thiol-substituted agarose. The number average chain length of the nascent DNA synthesized in one minute at 25 °C was 33 nucleotide residues, due to the chain-terminating action of 2′,3′-dideoxyATP. Several lines of evidence indicated that at least 90% of the DNA thus isolated carried a terminally phosphorylated RNA moiety at its 5′-end: (1) the nascent DNA was resistant to exonucleolytic degradation by spleen phosphodiesterase unless first hydrolyzed by strong alkali or ribonuclease; (2) the 5′-termini of nascent DNA could not be phosphorylated by polynucleotide kinase unless first treated with alkaline phosphatase or subjected to hydrolysis by strong alkali or ribonuclease; (3) alkaline hydrolysis of nascent DNA labeled with 32P at the 5′-end released unlabeled DNA with a free 5′-terminus and 32P-labeled ribonucleoside 3′,5′-bisphosphates; (4) ribonuclease degradation of similarly labeled material produced an unlabeled DNA-containing polynucleotide fraction and 32P-labeled ribo-oligonucleotides; (5) chromatography on dihydroxyboryl cellulose showed that the RNA moiety lacked a 3′-terminal cis-diol grouping (even after treatment with alkaline phosphatase) unless first subjected to the 3′-exonucleolytic action of bacteriophage T4 DNA polymerase. The sequence of the ribonucleotide chains was elucidated by end-group labeling with polynucleotide kinase and digestion with various ribonucleases. The ribonucleotide moiety was primarily three and four residues in length with the predominant sequence (pp)pApG(pC) 1–2pDNA. The possibility that it represents a primer for discontinuous DNA synthesis is discussed.

Involvement of outside DNA sequences in the major kinetic path by which EcoRI endonuclease locates and leaves its recognition sequence.

We have examined the kinetics of the interaction between endodeoxyribonuclease EcoRI (EC 3.1.23.13) and nine linear DNA fragments that range in size between 34 and 6,200 base pairs and contain the EcoRI site of plasmid pBR322 in a central location. The kinetic parameters governing both formation and decay of specific endonuclease . DNA complexes increase 8-fold with increasing chain length over this size range. In contrast, equilibrium competition experiments demonstrated that the intrinsic affinity of endonuclease for its recognition sequence is independent of DNA chain length over this range. Thus, DNA sequences outside the recognition site enhance the rate at which EcoRI endonuclease locates or leaves its recognition site without affecting the intrinsic thermodynamic parameters of site-specific interaction. These results are consistent with a facilitated diffusion mechanism for specific DNA site location by this enzyme.

Association kinetics with coupled three- and one-dimensional diffusion. Chain-length dependence of the association rate of specific DNA sites.

The effective target size of a specific binding site on a long polymeric chain can be considerably enhanced if the associating molecules slide along the chain in a one-dimensional diffusion. Such a mechanism has been proposed to explain the "faster than diffusion controlled" association of the lac repressor protein with the operator site on a long DNA chain. An important experimental tool for investigating the properties of rate-enhancing mechanisms of this type is now becoming available. In these experiments the DNA chain length is varied and the association rate of the investigated protein is measured. Previous attempts to describe theoretically how such one-dimensional sliding processes behave under those conditions were either oversimplified or connected with fundamental errors. We present here a theory for the association rate of proteins with a specific site on DNA molecules with variable lengths. We use a steady-state approach as do Schranner and Richter (Biophys. Chem. 8 (1978)47) but, in contrast, use boundary conditions which appear to be physically more reasonable.

Specific sequences in native DNA that arrest synthesis by DNA polymerase alpha.

The effect of the DNA sequence of a template on the progress of DNA polymerase alpha was determined at the resolution of single nucleotides by using single-stranded, circular phi X174 DNA as a template and unique phi X 174 ONA fragments terminally labeled at their 3'-ends as primers. Therefore, the amount of radioactivity observed was always proportional to the number and not the length of nascent DNA chains. Extension of a primer by alpha-polymerase revealed that 3'-ends of nascent DNA chains accumulated at specific arrest sites consisting of GC-rich sequences of 1-8 bases distributed nonuniformly along the template with intervening sequences of 0-140 bases. The precise location and composition of these sites were determined by DNA sequencing methods, but a consensus sequence was not detected. However, the same pattern of arrest sites recognized by DNA polymerase alpha from CV-1 cells also arrested alpha-polymerase from HeLa cells, calf thymus tissue, and phage T4 DNA polymerase. An exception was DNA polymerase I from Escherichia coli which was insensitive to arrest signals. Analysis of recombinant DNA templates containing phi X174 DNA inserted into M13 DNA revealed that arrest sites were defined exclusively by those sequences within 24 bases upstream and 140 bases downstream of the arrest point; long range interactions between template sequences were not involved. One of the strongest arrest sites was located in the first two nucleotides at the base of the stem on the primer-proximal side of a stable hairpin structure, suggesting that such structures arrest the polymerase. However, only 28% of all arrest site nucleotides were found in this position; the rest were up to 25 bases upstream from any computer-predicted hairpin. Therefore, all arrest sites cannot be defined by secondary structure alone, although proximity to secondary structures may amplify normal variations in the rate of DNA elongation caused by primary sequences.

Growth of Replicating DNA Chain in Adult and Old Rat Spleen

The rate of growth of the DNA chain length was found to decrease in rat spleen with aging. This can be substantiated by the slowing down of transformation of old spleen 3H-labeled DNA with low molecular weight to that with high molecular weight as labeling time increases. At the same time, the intensity of the DNA total synthesis is higher in old spleens than in adult ones. This may result from an increase of mitotic activity in rodent spleen with aging. In its turn, the increased mitotic activity may represent an adaptive reaction responsible for maintaining the number of immunocompetent cells which tend to decrease with aging. The slowing down of the newly formed DNA chain growth may lead to the decreased rate of stabilization of the secondary structure of replicating DNA in old spleen.

38] Fractionation of low molecular weight DNA or RNA in polyacrylamide gels containing 98% formamide or 7 M urea

This chapter describes the techniques for fractionation of low molecular weight DNA or RNA in polyacrylarnide gels containing 98% formamide or 7 M urea. The chain length of small, single-stranded DNA or RNA molecules can be determined by electrophoresis in polyacrylamide gels containing 7 M urea or 98% formamide. Under the appropriate conditions, the electrophoretic mobility of these molecules is independent of base composition and secondary structure. Denaturing polyacrylamide gels containing 7 M urea or 98% formamide are used to purify nucleic acids because of the high resolution afforded by the technique and the ease and efficiency by which nucleic acids can be recovered from the gels. The chapter describes the procedures for preparing polyacrylamide gels containing 7 M urea or 98% formamide and a procedure for extracting nucleic acids from these gels.

DNA chain length markers and the influence of base composition on electrophoretic mobility of oligodeoxyribonucleotides in polyacrylamide-gels.

The specific influence of the four nucleobases on electrophoretic mobility of oligodeoxyribonucleotides in polyacrylamide-gels under denaturing and nondenaturing conditions has been investigated using homooligomers from the four deoxyribonucleotides as chain length standards. Homooligomers of same chain lengths exhibit remarkable differences in mobility. Specific retardation of any other oligonucleotide investigated was found to be mainly dependent on base composition but not on sequence. A simple procedure is presented for calculating mobilities relative to the standards on denaturing gels. This allows a reliable identification of oligonucleotides on acrylamide-gels by exact chain length determination with respect to base composition and furthermore a detailed interpretation of complex reaction mixtures. The homooligomers also show the same differences in mobility on nondenaturing gels. The significance of this effect for strand separation is discussed.