Detection of point mutations in human DNA by analysis of RNA conformation polymorphism(s).
RNA molecules were found to separate into numerous metastable conformational forms upon non-denaturing gel electrophoresis. The equilibration of the conformations was accelerated by heating or mild denaturing conditions. Single-base substitutions in the sequence of the RNAs caused changes 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 T7 RNA polymerase. The method is rapid, simple, amenable to non-radioactive visualization and was successful in several cases when DNA single-strand conformational polymorphism analysis (Orita et al. (1989) Genomics 5, 874-879) failed to detect the point mutation.
Danenberg
P V
PV
Kenneth Norris Jr. Comprehensive Cancer Center, University of Southern California School of Medicine, Los Angeles 90033.
Horikoshi
T
T
Volkenandt
M
M
Danenberg
K
K
Lenz
H J
HJ
Shea
L C
LC
Dicker
A P
AP
Simoneau
A
A
Jones
P A
PA
Bertino
J R
JR
D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000235:genetics*; D004586:Electrophoresis; D016158:Genes, p53 / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D012313:RNA / Q000737:chemistry*; D013696:Temperature; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics
D012313:RNA; D004247:DNA; D013762:Tetrahydrofolate Dehydrogenase