A one-step DGGE scanning method for detection of mutations in the K-, N-, and H-ras oncogenes: mutations at codons 12, 13 and 61 are rare in B-cell non-Hodgkin's lymphoma.
Mutations in the N-, K-, and H-ras genes are key events in the process of carcinogenesis of many human cancers and may serve as important targets for therapeutic intervention. We developed a simple diagnostic method that in one step and within 5 hr determines the mutational status of any of the 3 ras genes in a given tumor sample. The method combines polymerase chain reaction (PCR) with denaturing gradient gel electrophoresis (DGGE) and allows simultaneous mutation scanning of 6 regions covering "hot-spot" codons 12, 13 and 61 of the 3 ras genes. The sensitivity of the assay was demonstrated by the analysis of control mutations, either naturally occurring or created by site-directed mutagenesis. We further demonstrate that unambiguous identification of ras mutations can be achieved by heteroduplex analysis in denaturing gradient gels, circumventing sequence analysis. We applied the method to establish the mutational status of all 3 ras genes in 123 samples of B-cell non-Hodgkin's lymphoma. Altogether, one diffuse large B-cell lymphoma and one B-cell chronic lymphocytic leukemia (B-CLL) harbored a mutation (G12S and G12A, respectively) in the K-ras gene, and one B-CLL harbored a mutation (Q61R) in the N-ras gene. We therefore conclude that ras mutations only contribute rarely, if at all, to carcinogenesis in B-cell non-Hodgkin's lymphoma.
Nedergaard
T
T
Department of Tumor Cell Biology, Danish Cancer Society, Copenhagen.
Guldberg
P
P
Ralfkiaer
E
E
Zeuthen
J
J
D001483:Base Sequence; D003062:Codon; D004273:DNA, Neoplasm / Q000737:chemistry; D005091:Exons; D011905:Genes, ras; D005821:Genetic Techniques; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000235:genetics; D016393:Lymphoma, B-Cell / Q000235:genetics*; D016403:Lymphoma, Large B-Cell, Diffuse / Q000235:genetics; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D017354:Point Mutation; D016133:Polymerase Chain Reaction
D003062:Codon; D004273:DNA, Neoplasm