Detecting base pair substitutions in DNA fragments by temperature-gradient gel electrophoresis.

A vertical gel electrophoresis apparatus is described which can distinguish DNA fragments differing by single base pair substitutions. The system employs a homogenous polyacrylamide gel containing urea-formamide and a temperature gradient which runs either perpendicular or parallel to the direction of electrophoresis. The temperature-gradient system simplifies several features of the denaturant-gradient system (1) and is relatively inexpensive to construct. Eight homologous 373 bp DNAs differing by one, two, or nine base pair substitutions were examined. DNA electrophoretic mobility changed abruptly with the temperature induced unwinding of DNA domains. GC to AT substitutions at different locations within the first melting domain, as well as an AT to TA transversion were separated with temperature gradients parallel to the electrophoretic direction. The relative stabilities of the DNAs observed in the gels were compared to predictions of DNA melting theory. General agreement was observed however complete correspondence was not obtained.     

Polyethylene glycol derivatives of base and sequence specific DNA ligands: DNA interaction and application for base specific separation of DNA fragments by gel electrophoresis.

Various base pair specific DNA ligands comprising a phenyl phenazinium dye, a triphenylmethan dye and Hoechst 33258 were covalently bound to polyethylene glycol (PEG) via ester or ether bonds. The DNA interactions of the PEG derivatives formed were shown to exhibit the same base pair specificity as the parent compounds. Since the PEG chains thus bound to the DNA could be expected to increase drastically the frictional coefficient of the DNA, the PEG derivatives were used for base specific DNA separations in agarose and polyacrylamide gel electrophoresis. The procedures, which do not require any special techniques, are described in detail. The resolution observed in agarose gels allows one to separate equally sized DNA fragments differing as little as 1% in base composition at mean travel distances of about 10 cm. Examples of gels showing the base compositional heterogeneity of restriction fragments obtained from lambda DNA, E. coli DNA and calf thymus DNA are given.     

Resolution ofFusarium sporotrichioides proteins by two-dimensional polyacrylamide gel electrophoresis and identification by sequence homology comparison in protein data base

Proteins fromFusarium sporotrichioides M-1-1, a T2-toxin-producing strain, were separated by two-dimensional polyacrylamide gel electrophoresis. One thousand two hundred and forty-four protein spots were resolved and 103 protein spots were subjected to N-terminal sequencing. Fifty-eight protein spots were sequenced and 48 proteins were observed to have blocked N termini. Forty out of 58 sequenced proteins were identified by homology search against the PIR protein sequence data base and protein superfamily data base, while the residual 18 sequences were not identified. Twenty-seven of the N-terminal-blocked proteins were subjected to mild anhydrous hydrazine vapor deblocking. Twenty-four spots were not deblocked indicating the presence of acyl groups at the N termini, while 3 proteins were deblocked showing the blocked group to be pyrroglutamyl carboxylic acid residues. The results can provide a more global view of cellular genetic expression than any other technique. The created data may offer a unique opportunity to link information with DNA sequence data.     

A theoretical method to predict DNA permutation gel electrophoresis from the sequence.

The gel electrophoretic permutation assays of DNA fragments experimentally investigated by different authors were theoretically reproduced using our theoretical model of sequence-dependent curvature. The general pattern of agreement obtained suggests that our method can be usefully adopted as an alternative to the experimental assay, in particular where the lack of a sufficient number of unique restriction sites in the fragment prevents the correct localization of the main bend site.     

Resolution of DNA topoisomerase II by two-dimensional polyacrylamide gel electrophoresis and western blotting.

Eukaryotic DNA Topoisomerase II (Topo II) has been studied using high-resolution two-dimensional polyacrylamide electrophoresis (2D-PAGE) and immunodetection of resolved proteins using specific antisera (Western blotting). Traditional methods of 2D-PAGE failed to resolve Topo II and neither nonequilibrium nor equilibrium pH gradients allowed Topo II to enter the first dimension gel. Exhaustive nuclease digestion and alternate protein solubilization strategies also produced negative results. We have developed altered first dimension pH gradient profiles and employed a more aggressive protein solubilization procedure which resulted in the resolution of Topo II. The 170-kDa polypeptide focuses with an apparent isoelectric point of approximately 6.5.     

Peptide mapping of protein bands from polyacrylamide gel electrophoresis by chemical cleavage in gel pleces and re-electrophoresis

A simple method has been developed for peptide mapping of protein bands obtained by polyacrylamide gel electrophoresis. The procedure is based on selective acid hydrolysis of aspartyl-prolyl bonds which occur in proteins with an average frequency of 1 per 400 amino acid residues. A gel piece containing the protein to be analyzed is soaked with 75% formie acid. For the subsequent incubation at 37°C for 24 h the gel piece is immersed in liquid paraffin. After removal of formic acid by lyophilization the gel piece is rehydrated in buffer and placed into the sample well of a second polyacrylamide gel on which the generated peptides are electrophoretically separated.     

Detection of a single base exchange in PCR-amplified DNA fragments using agarose gel electrophoresis containing bisbenzimide-PEG.

Using PCR fragments of known sequences derived from isolates of two related fungal species, simple submarine electrophoresis in agarose gels containing a bisbenzimide-PEG conjugate (H.A.-Yellow) has been shown to be capable of distinguishing DNA fragments 567 bp long which differ by as little as a single base change. However, only changes affecting bisbenzimide binding sites (which consist of at least four consecutive A/T bases) alter mobility; other changes are ineffective. A second ligand (H.A.-Red) with high G/C specificity is suggested which may be as effective in detecting other sequence changes.     

Drosophila topoisomerase II double-strand DNA cleavage: analysis of DNA sequence homology at the cleavage site.

In order to study the sequence specificity of double-strand DNA cleavage by Drosophila topoisomerase II, we have mapped and sequenced 16 strong and 47 weak cleavage sites in the recombinant plasmid p pi 25.1. Analysis of the nucleotide and dinucleotide frequencies in the region near the site of phosphodiester bond breakage revealed a nonrandom distribution. The nucleotide frequencies observed would occur by chance with a probability less than 0.05. The consensus sequence we derived is 5'GT.A/TAY decrease ATT.AT..G 3', where a dot means no preferred nucleotide, Y is for pyrimidine, and the arrow shows the point of bond cleavage. On average, strong sites match the consensus better than weak sites.     

An accurate method for estimating sizes of small and large plasmids and DNA fragments by gel electrophoresis

Several regression methods were tested for estimating the sizes of a wide range of plasmids (1.37-312 MDa) and restriction fragments (2.2-14.2 MDa) by agarose gel electrophoresis. The most accurate and least variable method was the multiple regression of log10 molecular size against log10 relative mobility and the reciprocal square root of the relative mobility. This method gave a good fit to all the data with low percentage errors of the molecular size estimates (less than or equal to 3.0 +/- 1.5%). It is suggested that with this method the molecular size of unknown plasmids can be accurately estimated using the plasmids from Escherichia coli V517 and E. coli IR713 as standards.     

DDGGE, a simplified denaturing gradient gel electrophoresis method for the detection of sequence variations in PCR fragments

The need for rapid analysis of sequence variations in PCR fragments of the same length is increasing in medical diagnostics and environmental studies. Therefore a modified denaturing gradient gel electrophoresis (DGGE) method was developed in which mixed PCR fragments of 1,500 bp could be analysed on a conventional DNA sequencing gel apparatus. In addition, PCR primers without long GC-clamps could be used to amplify the target genes. © Rapid Science Ltd. 1998