Computational simulation of DNA melting and its application to denaturing gradient gel electrophoresis.

Publisher Summary This chapter discusses the application of computational simulation of deoxyribonucleic acid (DNA) melting for denaturing gradient gel electrophoresis. Calculation of the theoretical pattern of thermal stability of DNA molecules of known sequence, together with calculation of the expected changes in electrophoretic mobility in gels under denaturing conditions, is the first useful step toward searching for sequence changes by means of denaturing gradient gels and in designing a gradient gel for the preparative isolation of mutants after intensive mutagenesis. The calculations permit a reasonably reliable test of the experimental prospects for new hypotheses and experimental designs. They show, for example, that about 50–70% of all possible single base changes that might occur within the human β-globin gene cluster would be detectable using the denaturing gradient system after the restriction fragments of the genome are hybridized with labeled, single strands corresponding to the same fragments of the normal sequence. The calculations are easy to execute for any sequence on a digital computer. They provide an indication of the regions within the sequence where base changes are likely to be detected, the magnitude of the effects that can be expected, and guidance as to the choice of restriction sites for fragmentation. The theoretical calculation is sufficiently reliable to serve as a means for the evaluation of some thermodynamic parameters relevant to melting from gel data and to signal anomalous properties. The chapter discusses theoretical treatment of the helix-random chain transition for complex sequences, the melting map program MELT, the mobility program MU, and so on. The melting calculation is useful in several different applications. The most elementary result is a description of the probability that each base is helical (or nonhelical) at specified temperatures.