PubMed · 7042796
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M H Johnson, L F Broering, W M Gooch. 1982. Fractured denture repairs.. https://doi.org/10.14219/jada.archive.1982.0293
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A rapid analysis of single-strand conformation polymorphism (SSCP) by capillary electrophoresis with laser-induced fluorescence (CE-LIF) detector was developed using a short-chain, linear polyacrylamide (PA) as sieving medium. Capillary filling of this low-viscosity medium and medium replacement were carried out by commercial capillary electrophoresis instruments. The approach was successfully applied to detect the C677T mutation of methylenetetrahydrofolate reductase gene. The influences of factors such as the concentration of polymers, voltage, temperature, and additives on the SSCP analysis were systematically investigated. Using 6% PA sieving medium and high electric field, four strands were resolved within 11 min in a DNA sample heterozygous for the C677T mutation, and a characteristic pattern was apparent for each of the three genotypes. When using multiple injection mode, the average analysis time per sample was reduced to about 4 min. In conclusion, our results indicate that CE-LIF may be an alternative to conventional SSCP analysis based on slab gel electrophoresis for the detection of genetic mutations. The technique is simple and rapid and is well suited to analysis of large numbers of clinical samples.
The gel electrophoresis mobility shift assay is widely used for qualitative and quantitative characterization of protein complexes with nucleic acids. Often it is found that complexes persist within electrophoresis gels for much longer than expected on the basis of their free-solution lifetimes. Volume exclusion, direct interaction with gel matrices and the reduction of water activity by the gel have been proposed as mechanisms enhancing the stability of complexes during electrophoresis. We have used the well-characterized interaction of the E. coli cyclic AMP receptor protein (CAP) with lactose promoter DNA to test these proposals. We found that the activity of water within polyacrylamide gels differs little from that of the buffer in which they were cast and that the dependence of the dissociation rate constant on water activity is too small for osmotic stabilization to contribute significantly to the lifetimes of CAP-DNA complexes. In addition, we found that a cross-linked gel matrix is not required for the stabilization of CAP-DNA complexes, that comparable stabilization is produced by three dissimilar polymers (linear polyacrylamide, dextran and polyethylene glycol), and that these polymers stabilize complexes more effectively than equivalent weight concentrations of their cognate monomers. While these results challenge the notion that direct interaction with the gel matrix contributes to the stability of protein-DNA complexes, they are all features expected of excluded volume mechanisms.
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.