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.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Research Project (R01)
Project #
5R01HG000345-14
Application #
2674199
Study Section
Special Emphasis Panel (ZRG2-GNM (03))
Program Officer
Graham, Bettie
Project Start
1987-09-01
Project End
2000-11-30
Budget Start
1998-09-01
Budget End
2000-11-30
Support Year
14
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Massachusetts Institute of Technology
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
City
Cambridge
State
MA
Country
United States
Zip Code
02139
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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