Studies on DNA melting are proposed that will lead to a novel method for comprehensive detection of sequence variants in human genes. The method differs from current procedures in offering rapid, readily automatable, reliable, and fully exhaustive indication of polymorphisms and mutations. The approach, like that of denaturing gradient gel electrophoresis, depends on melting theory and on the perturbation of the melting temperature of DNA by the presence of single-base (or larger) sequence changes or mismatches; but electrophoresis is replaced by fluorescence. In freezing detection from electrophoresis, the technique will permit rapid, simultaneous scrutiny of many small samples, a substantial length of sequence in each. The project will consist of the following efforts: (1) introduction of fluorescence as an indicator of DNA melting; (2) melting observation of DNA bound to a silica surface; (3) analysis of the distribution of thermal stability in a microbial gene using a large set of mutants; (4) rigorously testing melting theory with those results; (5) applying the detection system to detection of mutants in human Y chromosome genes (in collaboration with D. Page); and (6) analysis of the dependence of the domain structure of DNA on components of its environment - ions, etc. When incorporated into subsequent engineering development, this approach can be expected to effect a major change in the feasibility of large-scale mutation analysis and screening.
| Shiraishi, M; Oates, A J; Li, X et al. (1998) The isolation of CpG islands from human chromosomal regions 11q13 and Xp22 by segregation of partlymelted molecules. Nucleic Acids Res 26:5544-50 |
| Michikawa, Y; Hofhaus, G; Lerman, L S et al. (1997) Comprehensive, rapid and sensitive detection of sequence variants of human mitochondrial tRNA genes. Nucleic Acids Res 25:2455-63 |
| Shiraishi, M; Lerman, L S; Sekiya, T (1995) Preferential isolation of DNA fragments associated with CpG islands. Proc Natl Acad Sci U S A 92:4229-33 |
| Blanchard, B J; Park, T; Fripp, W J et al. (1993) A mitochondrial DNA deletion in normally aging and in Alzheimer brain tissue. Neuroreport 4:799-802 |
| Keats, B J; Pollack, M S; McCall, A et al. (1991) Tight linkage of the gene for spinocerebellar ataxia to D6S89 on the short arm of chromosome 6 in a kindred for which close linkage to both HLA and F13A1 is excluded. Am J Hum Genet 49:972-7 |
