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Nucleic Acids Research, 1992, Vol. 20, No. 3 573-579
© 1992

MOLECULAR BIOLOGY

Detection of point mutations in human DNA by analysis of RNA conformation polymorphism(s)

Peter V. Danenberg, Tetsuro Horikoshi, Matthias Volkenandt1, Kathleen Danenberg, Heinz-Josef Lenz, Luke C.C. Shea, Adam P. Dicker1, Anne Simoneau, Peter A. Jones and Joseph R. Bertino1

Kenneth Norris Jr. Comprehensive Cancer Center, University of Southern California School of Medicine Los Angeles, CA 90033 1Memorial Sloan-Kettering Cancer Center New York, NY 10021, USA

Received September 9, 1991. Revised February 3, 1992. Accepted February 3, 1992.

RNA molecules were found to separate into numerous metastable conforimatiional forms upon non-denaturing gel electrophoresis. The equilibration of the conformations was accelerated by heating or mild denaturing conditions. Single-base substitutions in the conformational patterns, including mobility shifts of major and minor conformations, appearance of new conformations and loss of other conformations. This sequence-dependent RNA conformational polymorphism was used to detect point mutations in p53 and, dihydrofolate reductase genes. Sense and anti-sense RNA strands corresponding to the same segment of the p53 gene gave entirely different conformational patterns. To generate the RNA, short regions of the target genes (up to about 250 bp) were amplified by the polymerase chain reaction and the resulting DNA segments transcribed to RNA by 17 RNA polymerase. The method is rapid, simple, amenable to non-radioactive visualization and was successful in several cases when DMA single-strand conformational polymorphism analysis (Orita et al. (1989) Genomics S, 874–879) failed to detect the point mutation.


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