Novel method of detecting single base substitutions in RNA molecules by differential melting behavior in solution.
We have developed a quick and reliable way of detecting point mutations in RNA molecules. This method involves melting RNA-RNA heteroduplexes of varying lengths in a series of tubes containing a stepwise salt or formamide gradient, followed by polyacrylamide gel electrophoresis to distinguish between single- and double-stranded RNA molecules. The manipulations required are technically simple, and the method is sensitive enough to detect destabilization of the highest melting domain of a dsRNA duplex by a single base mismatch. When this method is used in parallel with denaturing gradient gel electrophoresis, which detects point mutations in low-melting domains of duplexes, it should now be possible to rapidly screen for mutations located throughout the length of any RNA molecule whose wild-type sequence is known.
Smith
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FI
Mount Sinai School of Medicine, Department of Microbiology, New York, New York 10029.
Latham
T E
TE
Ferrier
J A
JA
Palese
P
P
D001482:Base Composition; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005814:Genes, Viral; D009980:Influenza A virus / Q000235:genetics*; D009154:Mutation; D009691:Nucleic Acid Denaturation; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D012367:RNA, Viral / Q000235:genetics*; D014158:Transcription, Genetic
D004279:DNA, Viral; D012367:RNA, Viral