Mark M. Garner

Effect of Z-DNA on nucleosome placement

Histone octamers were reconstituted on plasmids carrying the alternating nucleotide sequence (G-C)15. The plasmids, radioactively labeled at one of two neighboring sites near the (G-C) insert, were digested with micrococcal nuclease. Nucleosome core particles were isolated and the monomer DNA subjected to restriction analysis. Quite different results are obtained if the reconstitution is carried out with relaxed plasmids, in which the (G-C) insert is in the B form, or with supercoiled plasmids, where it is in the Z form. With supercoiled plasmids, there is a marked reduction (compared with relaxed plasmids) in the abundance of labeled monomers, the result of a large decrease in core particles carrying any (G-C) sequence. Some core particles formed on supercoiled (Z) plasmids are positioned either just outside the (G-C) sequence, or with the sequence occupying the terminal position within the core particle. In contrast, monomers obtained from relaxed plasmids incorporate the (G-C) sequence in the B form more or less randomly in the interior of the core particle; species showing discrete positioning make only a minor contribution. We conclude that DNA in the Z form cannot be incorporated within core particles, except at their termini, and that a transition from the B to the Z form in vivo might result in a significantly altered local placement of nucleosomes.

Use of a DNA toolbox for the characterization of mutation scanning methods. I: construction of the toolbox and evaluation of heteroduplex analysis.

A systematic characterization of the effects of important physical parameters on the sensitivity and specificity of methods in searching for unknown base changes (mutations or single nucleotide polymorphisms) over a relatively long DNA segment has not been previously reported. To this end, we have constructed a set of molecules of varying G+C content (40, 50, and 60% GC) having all possible base changes at a particular location — the “DNA toolbox”. Exhaustive confirmatory sequencing demonstrated that there were no other base changes in any of the clones. Using this set of clones as polymerase chain reaction (PCR) templates, amplicons of various lengths with the same base mutated to all other bases were generated. The behavior of these constructs in manual and automated heteroduplex analysis was analyzed as a function of the size and overall base content of the fragment, the nature and location of the base change. Our results show that in heteroduplex analysis, the nature of the mismatched base pair is the overriding determinant for the ability to detect the mutation, regardless of fragment length, GC content, or the location of the mutation.

Effect of gelation conditions on the gel structure and resolving power of agarose-based DNA sequencing gels.

The effect of gelation conditions on the structure and resolving properties of agarose‐based DNA sequencing gels were studied. Gels that were formed by rapid cooling gave separation patterns with sharp, well‐defined bands, and resolved base repeats that were not separated on gels which were allowed to cool slowly. Analyses by electron microscopy indicated that the rapidly cooled gels have a homogeneous pore structure with a smaller average pore size than gels that were cooled slowly. The two types of gels also differ in optical clarity, and have significant differences in their melting characteristics as measured by differential scanning calorimetry.

Use of DNA toolbox for the characterization of mutation scanning methods. II: Evaluation of single-strand conformation polymorphism analysis

Single‐strand conformation polymorphism (SSCP) is one of the most commonly used methods for searching for unknown base changes (mutations). In order to characterize systematically the effects of important physical parameters on the sensitivity and specificity of SSCP, we used the DNA toolbox constructed as described in the companion paper [2]. Using this set of DNA molecules as polymerase chain reaction (PCR) templates, amplicons of various lengths with the same base, mutated to all other bases, were generated. The behavior of these constructs in manual and automated SSCP was analyzed as a function of the size, overall base content of the fragment, nature and location of the base change, and the temperature and pH of electrophoresis. Our results demonstrate that all of these variables interact to determine the rate of detection of single‐base changes, with the GC content being the predominant determinant of detection sensitivity.

Injection bias of DNA fragments in capillary electrophoresis with sieving

The relative amounts of DNA fragments in a mixture injected into the capillary by electromigration or hydrodynamically by pressure were compared. Even if the electrophoretic mobilities of DNA fragments with different sizes are the same in a free solution in the sample vial, the size bias is brought about by the different mobilities in a sieving medium and by the electroosmosis. The experiments were performed in capillaries filled with a solution of liquified agarose, a replaceable sieving medium. The experimental results were compared with a theoretical model.

Computer-aided analysis of DNA curves on transverse gradient gels

Transverse pore gradient polyacrylamide gel electrophoresis of DNA restriction fragments was used to generate gel patterns describing migration distance as a function of gel concentration (Ferguson curves). These Ferguson curves were digitized, traced and analyzed with the aid of a personal computer. The traced curves were plotted semi-logarithmically and the plots were subjected to least-squares linear regression analysis to yield values of the slope (KR) and the intercept at %T = 0 (YO). These values are highly precise since they are based on approx. 100 measurements per curve. The computerized method reduces the errors due to manual measurements of migration distances and is time and labor saving. The method is still limited to intra-experimental comparison of Ferguson curves, since it does not as yet comprise a determination of gel concentration. At present, curve tracing remains semi-automated, requiring manual intervention when Ferguson curves cross or approach one another. Potentially, the importance of the computerized analysis of transverse pore gradient gels lies in the rapid quantitative interpretation of Ferguson curves for detection of anomalously migrating DNA species. Potentially, that application provides a more sensitive and informative mode of detection than either the mere visual observation of crossing Ferguson curves or of a shift in mobility at a single gel concentration.