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Sequence-determined DNA Separations.

L. Lerman, S. Fischer, I. Hurley, K. Silverstein, N. Lumelsky
Published 1984 · Biology, Medicine

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The variation in electrophoretic mobility of DNA under conditions of marginal helix stability provides a useful means for investigation of the relation between the helix-random chain transition and base sequence in natural DNA and a powerful procedure for separation of DNA molecules according to sequence. The use of statistical mechanical theory for analysis of the transition equilibria together with new, simplified theoretical considerations on the effect of strand unravelling on mobility have shown that the gel behavior is predictable for known sequences. A number of the distinctive consequences of the theory and their correspondence with the properties of real molecules have been demonstrated. These include the extremely close cooperative linkage of large blocks of bases into domains, the existence of sharp boundaries between domains, the major role of nearest-neighbor interaction in determining stability, the dependence of domain structures on neighboring and more remote sequences, and the depression of domain melting temperature if the sequence lies at the end of a molecule. New and unusual applications derive from the possibility of separating DNA molecules by properties of their sequence. Exceedingly complex mixtures, such as the sum of all fragments produced by the action of a sixbase specific restriction endonuclease on a complete bacterial genome, can be resolved completely. Additional inserted sequences are easily discerned. The difference of a single base pair in a molecule permits detection and isolation of mutant sequences. The need for full sequential analysis of long molecules for characterization of mutants can be reduced by localizing a change within a small fragment.

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