Identification of mutations by RNA conformational polymorphism "bar code" analysis.
DNA single-strand conformational polymorphism (SSCP) analysis is widely used for detection of point mutations in clinical specimens. Performing SSCP analysis with cRNA instead of DNA has been shown to improve mutation detection frequency. RNA can exist in numerous meta-stable conformations, which appear as patterns of bands on nondenaturing electrophoresis gels. Single base mutations can cause not only mobility shifts of major bands, but also loss of some conformations and appearance of new conformations. Unique RNA SSCP patterns associated with specific base sequences in many cases allow visual identification of point mutations. However, in some cases, the RNA SSCP pattern of a single base change in a sequence is not sufficiently different for a positive identification of the mutation. Improvement in the detection capability of RNA SSCP was obtained by adding 3'-deoxynucleotides to the transcription reaction. The presence of chain-terminating nucleotides in the transcription reaction formed numerous new RNA fragments, thereby generating complex band patterns ("bar codes") unique to each RNA sequence. This method was applied to analyzing p53 mutations in patients with colon cancer.
Lenz
H J
HJ
USC/Norris Comprehensive Cancer Center, Los Angeles, California 90033, USA.
Danenberg
K D
KD
Schnieders
B
B
Banerjee
D
D
Bertino
J R
JR
Leichman
L
L
Danenberg
P V
PV
D001483:Base Sequence; D003110:Colonic Neoplasms / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D016158:Genes, p53; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D018807:Polymorphism, Single-Stranded Conformational; D012313:RNA / Q000737:chemistry*; D014158:Transcription, Genetic
D012313:RNA